FAMRI 2003-2004 Funding Cycle Awardees

Clinical Innovators

• Andrew P. Lane
  Chronic sinusitis is an increasingly prevalent health care problem that affects greater than 30 million Americans each year. Although the underlying cause of this disorder is unknown, many factors have been demonstrated to promote or exacerbate the condition in susceptible individuals. Among these factors, air quality and airborne particulates such as tobacco smoke have been identified to play potentially important roles in the pathogenesis of chronic sinonasal inflammation. Long-term exposure to chemicals and other irritants stimulates nasal immune defense mechanisms, which can lead to the establishment of a chronic inflammatory state. It is well recognized that chronic sinusitis is common in the flight attendant population, although the relationship of this problem to the airliner cabin air environment is largely unknown. This proposal seeks to study the effects of specific elements of the airliner cabin environment on the production of pro-inflammatory mediators by sinonasal epithelial cells. In Aim #1, we will culture primary human sinonasal epithelial cells at the air-liquid interface and expose them to humidity, air pressure, and oxygen concentrations associated with in-flight airliner cabins. The expression of the pro-inflammatory cytokines TNF-a, IL-1b, interleukin 6, and interleukin 8 will be assessed before and after prolonged growth under these conditions. The expression of mucins and components of the complement system will also be examined. In Aim #2, we will study how the ambient conditions of the airline cabin environment impacts the epithelial inflammatory response to airborne chemical and microbial agents found in cabin air. Specifically, we will apply sidestream tobacco smoke, aircraft systems contaminants, carbon dioxide, ozone, and fungi to epithelial cell cultures in separate experiments. The expression of the same cytokines and proteins examined in Aim #1 will be assayed after exposure to these agents with or without the presence of cabin air conditions. Finally, in Aim #3 we will compare epithelial cells obtained from control subjects to those from patients with chronic sinusitis to test the hypothesis that chronic sinusitis leads to an increased inflammatory response to cabin air conditions. Through this research, it is hoped that some pathways leading to the development of sinusitis in the flight attendant population may be elucidated. A better understanding of the basic pathophysiology may ultimately lead to methods of treatment and prevention.
  
  
  
  
  
• Talissa Ann Altes, M.D., M.S.
  Hyperpolarized helium-3 gas is a new contrast agent that, when inhaled, permits magnetic resonance imaging (MRI) of the airspaces of the lung. By combining this technique with the established technique of diffusion MRI, it is possible to non-invasively image the size and morphology of the distal airspaces of the lung. Emphysema is characterized by destruction of alveolar walls and permanent enlargement of the airspaces distal to the terminal bronchioles, structural changes that can be detected by diffusion hyperpolarized helium-3 MRI. While experience with this new imaging technique is limited, it appears to be very sensitive to early emphysematous change in the lung. Active cigarette smoking is strongly associated with the development of emphysema. It seems plausible that second hand cigarette smoke could also cause emphysema; however, this association has been difficult to establish, likely due to the long latent period before the onset of symptoms and the difficultly in diagnosing early emphysema. Second hand smoke may be more likely to induce sub-clinical emphysema than clinical emphysema where sub-clinical emphysema is emphysematous change in the absence of symptoms or abnormal spirometry. Sub-clinical emphysema may cause morbidity by lowering respiratory reserve, and people with sub-clinical emphysema may be at greatly increased risk of developing clinical emphysema. By being a very sensitive test, diffusion helium MRI has the potential to detect early emphysematous change in the lung and, as such, be able to definitively determine whether an association exists between second hand cigarette smoke and pulmonary emphysema. The goal of the proposed study is to determine whether diffusion helium MRI is likely to detect such an association if one exists. We propose to determine whether diffusion helium MRI detects sub-clinical emphysematous change within the lungs of 40 never smokers who had a high lifetime exposure to second hand smoke. Next we will determine whether there is a dose response relationship between cigarette smoke exposure and the amount of emphysematous change in the lung on diffusion helium MRI by comparing the presence and severity of emphysematous change among three groups: 1) 40 never smokers with little exposure to second hand smoke, 2) 40 never smokers with a high lifetime exposure to second hand smoke, and 3) 25 active smokers. Lifetime second hand smoke exposure will be assessed by questionnaire.
  
  
  
  
  
• Douglas A. Arenberg, M.D.
  A critical feature distinguishing malignant transformation (on a cellular level) from tumorigenesis (at the tissue level) is angiogenesis, which is required to sustain tumor growth. In its 2004 Plans & Priorities for Cancer Research, the NIH recognized this distinction in stating ÒThe cancer cell is only part of the story in cancer development. As a cancer grows... it interacts with its surrounding environment. Mounting evidence now suggests that a dynamic interaction occurs between the cancer cell and its microenvironment.Ó This is especially true of the mechanism by which tumors achieve their angiogenic requirements. While tumor cells are capable of producing angiogenic factors, we and others have shown that normal ÒhostÓ cells are influenced to adopt a pro-angiogenic phenotype under the influence of the tumor microenvironment. One mechanism by which malignant cells influence host cells is the production of the cytokine macrophage migration-inhibitory factor (MIF). MIF is expressed in tumor cell lines, and may directly promote angiogenesis. We have found that MIF indirectly promotes angiogenesis in lung cancer by increasing the expression of other angiogenic factors. Additionally, MIF antagonizes the tumor suppressor p53. These properties of MIF will be exploited in this proposal to generate a transgenic mouse to facilitate the study of the angiogenic switch in lung cancer. The primary goal of this proposal is to support the generation of a transgenic mouse that permits precise control of the timing and nature of the angiogenic switch in the context of chemical induced tumorigenesis. We propose to generate a mouse strain that is double-transgenic for the murine MIF gene under a tetracycline-dependent promoter and the tetracycline operator on a lung specific (CC 10) promoter. Once this transgenic strain is established, we will subject them to urethane induced tumorigenesis. One of several long-term goals will be to determine whether induction of one angiogenic mechanism in developing tumors suppresses the development of other angiogenic mechanisms. We will use transgenic mice, as well as controls in urethane induced tumorigenesis experiments to determine the nature and control of the angiogenic switch in lung cancer. The development of this transgenic model will allow us to address an otherwise un-testable hypothesis; during tumor development, evolution of a given angiogenic strategy suppresses the development of other angiogenic strategies. If true, this has vital implications for therapies directed at inhibiting tumor angiogenesis.
  
  
  
  
  
• William P. Bennett, M.D., M.S.
  Background. Molecular signatures are a new tool for translational studies of cancer etiology and pathogenesis. Signatures emerged from observations linking some carcinogens to distinctive patterns of genetic damage. These patterns have been likened to electromagnetic spectra measured by chemists to identify unknown compounds. Just as emission profiles detect elements and composites, so can distinctive mutation "signatures" expose carcinogens. In this field, laboratory studies catalogue genetic damage carried by tumors, and common or distinctive or understandable features may constitute a molecular signature. Clear signatures can address questions of etiology, and the supporting data may (i) suggest strategies for early detection or prevention, or (ii) correlate with response, recurrence or survival.Current problem. Tobacco smoke produces distinctive p53 mutations in lung cancers. Although highly specific, this signature occurs in less than 20% of tumors, so a more reliable marker is needed. Recent evidence indicates that methylation and allelic deletion occur more frequently in lung cancers from smokers than nonsmokers. Hotspots have been reported as well. These observations suggest that a useful tobacco signature could emerge from distinctive patterns of methylation and deletion. Current problem. Tobacco smoke produces distinctive p53 mutations in lung cancers. Although highly specific, this signature occurs in less than 20% of tumors, so a more reliable marker is needed. Recent evidence indicates that methylation and allelic deletion occur more frequently in lung cancers from smokers than nonsmokers. Hotspots have been reported as well. These observations suggest that a useful tobacco signature could emerge from distinctive patterns of methylation and deletion. Principal Hypothesis. Tobacco smoke carcinogens promote methylation and allelic deletion in bronchial epithelial cells. Some of these genetic and epigenetic changes promote neoplasia, and some of the modifications persist in tumors that evolve later. Samples & Laboratory Methods. Lung cancers from smokers and nonsmokers were collected from epidemiologic studies; detailed smoking histories were collected by trained interviewers. Formalin_fixed tissues will be microdissected to provide genomic DNA for PCR_based assays. Data Analysis. Multivariate analysis will test coordinate genetic damage for power to discriminate prior exposure to tobacco smoke.
  
  
  
  
  
• Danny Bluestein, Ph.D.
  Cigarette smoke, whether inhaled in high doses by smokers, or inhaled in lower doses as secondhand smoke (SHS) by nonsmokers, greatly increases the risk of vascular disease and thrombosis. Enhanced function of the hemostatic system, in which platelets and endothelial cells (ECs) play a major role, is a significant underlying mechanism in cardiovascular disease and its progression. Platelets can be activated by myriad means, including exposure to abnormal flow stresses and exposure to exogenous stimuli like tobacco smoke. ECs are potent regulators of platelet behavior, but their normally antithrombotic role is reversed when they are stimulated, which can be induced directly by activated platelets. This project continues a previous study of the effects of cigarette smoke extracts on the susceptibility of platelets to thrombogenic activation under cardiovascular flow stresses conditions. In addition to studies deriving directly from the current project, we will extend it to ECs: both their direct activation by smoke extracts, and the combined effects of smoke on the platelets and ECs together in a circulation system. Combined activation of these two cell types initiates a potent inflammatory response, and we hypothesize that this may play a major role in the vascular response to cigarette smoke. Motivated by the recent marketing of low and zero nicotine cigarettes, we also propose to study nicotine effects. There is a substantial concern whether these cigarettes have any benefit. Conceivably, they carry additional cardiovascular risks. Nicotine, the major addictive ingredient in cigarettes, de facto is assumed harmful, but in fact its effect on platelet thrombogenicity is opposite from whole smoke: it desensitizes platelets to the effects of flow stresses, even at low levels. Studies examining this distinction will include analysis of the platelets of volunteers exposed acutely to (i) secondhand normal cigarette smoke; (ii) secondhand nicotine-free smoke; and (iii) pure nicotine (nicotine gum). ECs will be examined alone under flow stress in the presence of normal and nicotine-free smoke extracts and nicotine, measuring standard markers of EC activation. In the final phases of the project, platelets will be introduced and the combined effects of smoke and nicotine measured by determining the extent of platelet activation and adhesion to the EC surface. Platelet adhesion is generally accepted to be a key event in development of the inflammatory response, and we hypothesize that exposure to cigarette smokeÑbut perhaps not nicotineÑmay prove to be a major means of initiation of this process.
  
  
  
  
  
• Johnny L. Carson, Ph.D.
  The broad long-term objective of this proposed study is to determine the predictive value of human nasal epithelium for the early detection of pathohistology and assessment of risk of adverse respiratory health effects among individuals with quantifiable exposure to ETS. This effort will apply physiologic measurements of mucociliary and epithelial function performed in vivo and in vitro with parallel assessments of structural integrity of the conducting airway epithelium as a function of quantifiable ETS exposure history. These studies will employ an innovative approach, the air/liquid interface culture, which promotes optimal growth and maintenance of airway epithelium under in vitro conditions. This situation will facilitate an evaluation of the growth characteristics of a mucosal layer strategically significant as a primary respiratory defense mechanism that has been exposed to ETS in a non-experimental setting. In the initial stages of this project, exposure to ETS will be determined using standard techniques for the detection of nicotine, nasal nitric oxide levels will be determined, and the participants sorted into cohorts. Non-invasive nasal biopsies will be performed on each participant and the tissue subjected to measurement of ciliary beat frequency and assessment of ultrastructural organization of the epithelium with particular emphasis on characterization of ciliary defects, and pertinent immunohistochemical localization profiles determined. The biopsies will then used to establish air/liquid interface cultures and further assessments of epithelial growth and maturation will be performed. Studies of cultured epithelium will include comparative quantitation of epithelial phenotype, assessments of variability in gene expression, and susceptibility to viral infection. Data deriving from the different cohorts will be compared in order to identify associations with ETS exposure. These studies may have predictive value in the determination of the risk of developing adverse respiratory effects as well as early detection of pathologic conditions associated with ETS exposure.
  
  
  
  
  
• Scott W. Cousins, M.D.
  Age-related macular degeneration (AMD) is the most important cause of blindness in the elderly, affecting 14 million patients in the US alone. Most cases of blindness are caused by neovascular AMD, characterized by choroidal neovascularization (CNV), the invasion of abnormal new vessels into the retina from the subjacent vascular bed (choroid). Neovascular AMD shares many pathogenic features with other cardiovascular diseases, especially the contribution of macrophage infiltration and a strong association with cigarette smoking. This proposal will test the hypothesis that smoke-related nicotine directly acts upon circulating monocytes, or alters monocyte differentiation in the bone marrow, to induce partially activated monocytes in the blood. Upon recruitment to the eye with developing CNV (or other vascular beds), increased production of TNF-alpha or other relevant mediators by these partially activated monocytes will contribute to more severe neovascularization. The hypothesis will be tested in animals and humans. In mouse model of experimental CNV, the capacity of tobacco smoke or nicotine to increase severity of experimental CNV through macrophage activation will be determined. Also the capacity of tobacco smoke or nicotine to influence bone marrow myeloid progenitor function, resulting in their differentiation into activated monocytes long after smoking cessation, will be evaluated. In human AMD, a case-control study of smokers and nonsmokers will be done to evaluate the association between neovascular AMD, blood levels of cotinine and partial activation of circulating monocytes isolated from smokers and nonsmokers. To our knowledge, the ideas proposed in this application are innovative, especially the idea that smoking can induce long-lasting changes in bone marrow myeloid development resulting in persistent activation of circulating monocytes. If confirmed, analysis of blood monocyte activation status will serve as a biomarker for risk of progression of AMD. Importantly, all of this information will be directly applicable to other cardiovascular diseases associated with cigarette smoking.
  
  
  
  
  
• Jeanine D'Armiento, M.D., Ph.D.
  Active and passive smoking is common among individuals with asthma. Tobacco smoke has been shown to result in increased morbidity from asthma, possibly due to the injurious effect of tobacco smoke on airway inflammation and remodeling. While extensively studied in COPD, the effects of smoking on the airway inflammation in asthma have not been well defined. Smoking-induced release of mediators from airway cells in asthma may result in matrix deposition and collagen deposition, which may explain the accelerated decline in lung function reported in large longitudinal studies of asthmatic patients who smoke. In addition, smoking may modify the typical airway inflammatory indices that have been described in asthma in a fashion that leads to worse lung function, less reversibility of airflow obstruction and increased bronchial hyperresponsiveness. We propose to compare the inflammatory cytokine and protease/antiprotease profile in the airways of asthmatic patients exposed to active and/or passive smoking with that from unexposed asthmatics. These studies will allow us to gain insight into the mechanistic causes of increased morbidity and accelerated loss of lung function among asthmatics who are exposed to cigarette smoke. We expect to find differences in collagen synthesis and protease/antiprotease stoichiometry in a pattern that favors greater matrix deposition and increased inflammation among active and passive smokers compared with asthmatic not exposed to cigarette smoke. We also expect to demonstrate worsened quality of life, increased degree of fixed airway obstruction and bronchial hyperresponsiveness among asthmatic patients exposed to cigarette smoke, in a manner that correlates with sputum indices of airway remodeling. These studies will lay the groundwork for future work to understand the mechanism responsible for the deleterious effect of cigarette smoke in the airways. We hypothesize that asthmatic patients exposed to active or passive tobacco smoke have more severe airway inflammation and remodeling compared to asthmatics who are not exposed to smoke. Specific Aims: Measure and compare biomarkers of airway remodeling (metalloproteinases and their inhibitors) and inflammation (cytokine levels) in the induced sputum of mild and moderate asthmatic patients exposed to active tobacco smoke, passive tobacco smoke and no tobacco smoke. Compare reversibility of airflow obstruction, airway hyperresponsiveness and asthma quality of life among asthmatics exposed to active smoking, passive smoking and no smoking.
  
  
  
  
  
• Joel G. Fletcher, M.D.
  Several methods indirectly measure vessel stiffness (or distensibility) in the aorta currently. Abnormal aortic distensibility is thought to be related to the development of high blood pressure, to cardiovascular mortality in patients with high blood pressure, and to the rupture of abdominal aortic aneurysms. Recent advances in ECG-gated multidetector CT technology have made possible time-resolved imaging of the entire abdominal aorta, such that multiple three-dimensional datasets can be created over the time period of the cardiac cycle to show pulsatile motion of the abdominal aorta using a single CT scan. These advances have made direct measurement and quantification of regional aortic distensibility possible. The objective of our investigation is to optimize these new CT techniques, to show that ECG-gated multidetector CT can be used to quantitate regional aortic distensibility. These techniques can be used to assess risk of abdominal aortic aneurysm rupture and measure early large vessel stiffness. Specifically, we plan to develop algorithms to automatically measure aortic pulsatility from time_-resolved CT data, and test the hypothesis that aortic aneurysm distensibility is an independent risk factor for abdominal aortic aneurysm rupture or intervention. We will also develop and optimize techniques to measure aortic distensibility using unenhanced CT. We will recruit 100 patients who are unlikely to undergo immediate surgery for their abdominal aortic aneurysms, but who will have their aneurysms followed by CT. Time-_resolved ECG-gated multidetector CT angiography will be performed in these patients at baseline and at follow-up imaging over one year. We will develop and employ novel aortic distensibility software to measure regional aortic distensibility, create pulsatility maps of the aorta, and to quantitate maximum aortic distensibility associated with each abdominal aortic aneurysm. We will compare maximum pulsatility in each patientÕs aneurysm to outcome over the follow-up period (that is, rupture or intervention vs. no intervention) to determine if aneurysm distensibility does indeed predict a rupture or intervention. This information can be used to guide decisions future decisions regarding treatment of abdominal aortic aneurysms. Concurrently with this project, we will develop software to measure aortic distensibility in CT scans without intravenous contrast dye, so that aortic distensibility can be measured in other patient groups. Using specialized noise insertion algorithms, we will insert noise into raw CT data, mimicking CT scans performed at lower and lower radiation doses. We will consequently determine how to minimize radiation dose while achieving reproducible measurements for aortic distensibility.
  
  
  
  
  
• Yuman Fong, M.D.
  Long term exposure to cigarette smoke increases the risk of a number of malignancies including lung, bladder and head and neck cancers. Much investigation has therefore focused on screening methods for detecting cancer development in high risk individuals with the goal of finding cancers at an early and potentially curable stage. Cytologic analysis of sputum or urine is currently used in screening for lung and bladder cancers but is hindered by the difficulties in detecting the rare tumor cell within the background of vast numbers of non-cancerous cells. In recent years, investigators have genetically engineered the herpes simplex virus for killing of cancers. We hypothesized that these oncolytic herpes viruses, which are highly efficient and specific in infection and replication within tumor cells, may also be used for detection of cancers. The current proposal seeks to determine if NV 1066, a mutant herpes that carries a gene for Green Fluorescent Protein (GFP), can be used to treat sputum or urine specimens, and result in specific expression of GFP in tumor cells to aid in their detection. We have preliminary data indicating that NV 1066 can infect a wide panel of lung and bladder tumors, and that the resultant GFP expression allows detection of tumor at a concentration of less than 1 cancer cell per 1 million normal cells. This proposal seeks to further define the limits of detection of such a herpes-mediated fluorescent imaging technique. These studies also seek to optimize timing, viral dose, and specimen handling for this novel detecting technique. Furthermore, studies are proposed seeking to combine such fluorescent imaging with tradition cytologic techniques to further enhance sensitivity for tumor detection. These are, therefore, practical studies that seeks to optimize a novel technique for use in man to increase yield of an easily performed screening test, with the hope that use of herpes-mediated fluorescent imaging will in the future increase detection of potentially curable cases of cancer.
  
  
  
  
  
• Deborah Gentile, M.D.
  Strong epidemiological evidence now links childhood asthma/atopy with the development/persistence of T helper lymphocyte type 2 (Th2) cytokine immune responses during early childhood. However, the ontogeny of human cytokine immune responses and the environmental factors influencing their development have not been fully elucidated. Several lines of evidence support the hypothesis that environmental tobacco smoke (ETS) during early childhood induces/amplifies Th2 cytokine immune responses. Moreover, results from recent studies suggest that diminished IL-10 and/or IL-12 production by dendritic cells (DCs) is a mechanism by which this occurs. The long-term goals of this research are to 1) elucidate the mechanism by which ETS predisposes to the development/persistence of Th2 cytokine immune response and the development of childhood asthma/atopy, and 2) identify potential strategies to attenuate or prevent the development of childhood asthma/atopy. The specific aims of this proposal are to 1) characterize the ontogeny of human cytokine immune responses during early childhood, 2) investigate the effect of ETS during on these immune responses, and 3) to develop a model for investigating the relationship between ETS-induced immune responses and the development of childhood asthma/atopy. This cross-sectional cohort study will enroll 210 subjects, including 35 subjects in each of the following groups: non-ETS subjects aged 1-2 years, ETS subjects aged 1-2 years, non-ETS subjects aged 3-4 years, ETS subjects aged 3-4 years, non-ETS subjects aged 5-6 years, and ETS subjects aged 5-6 years. Blood will be obtained for immune studies and the quantification of serum cotinine levels. Data will be analyzed to determine whether ETS during early childhood alters the ontogeny of DC IL-12 and IL-10 production and Th2 cytokine responses. In addition to providing insight into the mechanism by which ETS may predispose to the development of childhood asthma/atopy, this work may result in the identification of potential strategies to attenuate or prevent the development of this disorder.
  
  
  
  
  
• Mark S. Gold, M.D.
  The health risks related to smoking tobacco products are well known. These risks are related to direct use of tobacco products and exposure to passive smoke, sidestream smoke, second hand tobacco smoke (SHTS). Smoking is the single most important preventable cause of death and disability in the U.S., causing 450,000 deaths annually to smokers and those exposed to SHTS. Because it is impossible to eliminate past exposure and difficult to police tobacco use in the home, it is desirable to be able to detect and quantify SHTS exposure during encounters with healthcare professionals. Screening for tobacco exposure, recommended by the Centers for Disease Control should become a standard addition to the primary care exam, an additional vital sign. While physicians are ideally positioned to screen for SHTS and SHTS related diseases, they may not be asking about it, especially if they lack competency in SHTS issues. A major objective of this project is to assess current practices in screening for and quantifying SHTS exposure, though the ultimate goal is to increase effective screening and awareness of the need for it. We will design, pilot test and implement an SHTS competency exam for physicians and medical students. A random sample of 300 physicians who specialize in Internal Medicine, Family Practice, OB/GYN, Pediatrics, Pulmonology, Cardiology and Oncology will be included nationally. If, as we suspect, screening is less than optimal and competency is lacking, we will develop training, remediation and continuing education materials. Our written CME program focused on SHTS will be mailed to 25,000 MDÕs nation-wide and will be made available with a self-scoring test engine on the UF CME website so that MDs throughout the country can access and learn. The SHTS materials will be applicable for a wide range of health science students and professionals and will have a national impact. A better understanding of SHTS will allow physicians to quantify disease risk, prepare them to screen for tobacco related illnesses and encourage them to offer advice to their patients.
  
  
  
  
  
• Ellen J. Hahn, DNS, RN
  The broad, long-term objective is to assess the relationship between reduction in exposure to secondhand tobacco smoke (SHTS) at the community level and health care outcomes associated with acute coronary syndromes and asthma. The specific aim is to evaluate the effects of a community initiative to reduce exposure to SHTS on cardiac and asthma outcomes including the rate of hospital and emergency department (ED) discharges, length of stay, and total hospital costs. Although many local communities have adopted measures to reduce exposure to SHTS, the tobacco-growing states lag behind in protecting workers from SHTS. The Lexington-Fayette (Kentucky) Urban County Government enacted a smoke-free law on July 1, 2003, making all public buildings smoke-free including, but not limited to, restaurants, bars, bowling alleys, bingo halls, laundromats, and racetracks. LexingtonÕs initiative is considered by the Americans for NonsmokerÕs Rights as one of the strictest local laws in the U.S. The effective date was September 29, 2003. However, at the time of the grant submission, the enforcement of the initiative was temporarily delayed due to a court injunction. The study will be a time series design using five 6-month time periods; three time points prior to and two time points after enforcement of the Smoke-Free Lexington initiative. The administrative data base (Comp Data) used to summarize and track all Kentucky hospital billing records will be accessed for all Lexington hospitals during each of the five time periods. Comp Data will be queried for hospital discharge diagnosis by DRGs and ICD-9 codes, length of stay, total hospital charges, dates of service, gender, age, and zip code of residence. Since Comp Data does not compile emergency department (ED) discharge information, each Fayette County hospital will provide all data fields of interest above for the ED discharge diagnosis by DRGs and ICD-9 codes. Based on the enforcement date of the community intervention in Lexington (projected November 1, 2003), the data collection time periods will be in 6-month time intervals from November 1 of a given year to April 30 of the following year. Multiple linear regression will be used to determine predictors for length of stay and total costs; predictors will include year, month, gender, age, and an indicator variable for whether the observation occurred before or after the initiative. For the number of events (hospital and ED discharges) per at-risk population, an incidence rate, Poisson regression will be used with the same predictors.
  
  
  
  
  
• Alexander M. Ishov, Ph.D.
  One of the most obvious causes of lung cancer is active or passive exposure to tobacco smoke. Tyrosine kinase receptors (TKR) have been implicated recently in the etiology of lung malignancy and are therefore important therapeutic targets. TKR c-met is a proto-oncogene with established functions in tumor invasiveness and metastasis. C-met is overexpressed in many types of tumors, including lung cancer, and is associated with bad survival prognosis. We have found that expression of c-met is repressed by Daxx, a ubiquitous transcriptional modulator. Hence, Daxx is a potential tumor suppressor and is an ideal target to study the physiological regulation of c-met in carcinogenesis, more specifically, in the lung cancer progression. Based on interactions of Daxx with several members of the histone deacetylase (HDAC) family and SWI/SNF-like protein ATRX, we hypothesize that Daxx might inhibit c-met transcription recruiting to the c-met promoter region histone deacetylation activity and/or DNA methylation/chromatin remodeling activity attributed to ATRX. Downregulation of Daxx or spontaneous mutation inactivating Daxx could release this repression leading to elevation of c-met expression. This event, in turn, could result in oncogenic transformation and tumor progression. The proposed project is to test for the tumor suppression function of Daxx using human lung cancer cell lines and produced mouse Daxx knockout system. Specifically, I plan to answer the following questions: 1. What is the mechanism of Daxx mediated repression of c-met promoter? I plan to examine the c-met promoter for the histone modification and DNA methylation pattern in Daxx-/- and Daxx+/+ cells. To search for alternative mechanisms of Daxx-mediated repression, I will screen for new Daxx-interacting proteins using Daxx complex isolation and yeast two-hybrid analysis. 2. What are the physiological consequences of c-met over-expression in the absence of Daxx? I will test whether over-expression of c-met affects the growth pattern, invasive capacity and tumor/metastases production of Daxx-/- cells. 3. What is the state of Daxx in the c-met over-expressing lung malignancies? I plan to analyze the correlation between the status of c-met and Daxx in lung cancer cells and tissue samples. The long-term goal of this application is to understand the molecular mechanisms of lung cancer progression studying Daxx mediated transcription repression of c-met proto-oncogene, to determine whether Daxx transcriptional activity is downregulated in human lung cancer and how novel cancer therapies can be implemented by enhancing Daxx-mediated repression.
  
  
  
  
  
• Francine L. Jacobson, M.D., MPH
  The long-term goal of the proposed research is to find a measurement that can be used for screening purposes, to predict COPD or impending complication from COPD from a lower technology CT scan such as might be available on a van that could be taken to remote locations. The health relevance of this project lies in the fact that COPD ranks fourth as the most common cause of death in the US and is increasing in both prevalence and mortality. Only about 15-20% of smokers develop clinically significant COPD as determined by assessment of pulmonary function [Sherrill, US Surgeon General]. Furthermore, it is unclear why some smokers develop mainly airway disease (chronic bronchitis) while others develop mainly parenchymal disease (emphysema). The reason for the great inter-individual variability in the response to smoking, and to other environmental factors such as occupational dust exposure and diet remains unclear. It is likely that genetic risk factors also play a role in the variable response to cigarette smoking and other environmental factors. The Specific Aims of the project are to: (1) Survey 1000 Veterans with already known smoking histories regarding passive smoke exposure to select 2 groups of 50, one with history of heavy tobacco smoking and one with heavy exposure to second hand smoke; (2) Assess the presence or absence of COPD based on objective structural measurements from high resolution CT scans and compare with the detection of COPD using standard measures of lung function such as pulmonary function tests; and (3) Compare the use of clinical and CT risk assessments between the groups exposed to active and passive smoke to determine the optimum method for the assessment of risk in those individuals exposed to second-hand smoke. The Research Design and Methods will include questionnaire to evaluate health status and exposure to second hand smoke, pulmonary function testing with spirometry, and multi-detector CT of the thorax. Radiologist observations made with the assistance of standard workstation tools, and several measures derived by image processing performed by computers without human interaction. These will be performed in accordance with the standard procedures in current use, similar to those elaborated in the literature cited. The particular goal of this study is to look at measurements that can be made accurately using the highest level of technology currently available with an eye to selecting the most meaningful measurement, or measurements, that can be made with lesser levels of technology. Our long-term goal is to find a measurement that can be used for screening.
  
  
  
  
  
• Jean Kim, M.D., Ph.D.
  Chronic sinusitis is a disease of the nasal and sinus passages that leads to severe nasal congestion, nasal blockage, nasal discharge, loss of the sense of smell, facial pressure and pain, and post-nasal drip. The disease is thought to involve inflammation of the soft tissue lining of the sinus cavities. In part, this lining is composed of a type of cells, which form a protective barrier, called epithelial cells, and specialized white blood cells, called T cells, which "coordinate" the inflammatory process. New studies from our laboratories suggest that the epithelial cells are able to communicate with the T cells because they have on their surface specialized molecules, called costimulatory molecules, that can direct T cells to function during inflammation. We have found increased levels of these specialized costimulatory molecules on the epithelium in the nasal passages of patients with sinusitis. We plan to test the idea that the epithelial cells become activated during sinus disease. To measure activation, we will monitor costimulatory molecule expression on the surface of the epithelial cells in patients with chronic sinusitis. We will compare costimulatory molecule expression on nasal epithelial cells from human subjects before and after an attack of sinus disease. We will also collect nasal epithelial cells from human subjects and grow them in cell culture for laboratory studies. We will examine the effect of factors that we think cause chronic sinusitis, such as the common cold virus, on levels of activation of these cultured cells. We believe that these studies will provide new insights into the details of the inflammatory processes that underlie chronic sinusitis.
  
  
  
  
  
• Landon S. King, M.D.
  Chronic bronchitis and sinusitis affect millions of American each year at a cost of billions of dollars. While the molecular pathogenesis of these disorders remains undefined, disruption of the airway surface layer and altered secretions are common to the clinical expression of both conditions. The extent to which alterations in the airway surface layer are cause or consequence is unclear, however changes in the volume or constitution of the surface liquid can negatively impact on mucous and mucociliary clearance, and attenuate the normal protective effects of airway antibacterial peptides. Aquaporins are water-specific membrane channel proteins that determine water permeability in a variety of tissues. In the airways, AQP5 is expressed in the apical membrane of the superficial epithelium and submucosal glands, sites where contribution to the airway liquid is predicted. Mice with deletion of AQP5 have reduced secretion from secretory glands including airway submucosal glands, as well as increased bronchoconstriction in response to methacholine. We observed EGF receptor-mediated hypertonic induction of AQP5 in lung epithelial cells, and cAMP-mediated regulation of AQP5 abundance and distribution. TNF (in vitro) and acute bacterial infections (in vivo) reduce AQP5 abundance. We observed markedly reduced AQP5 in the lung epithelium of humans with emphysema, particularly adjacent to inspissated mucus. We believe these observations strongly indicate that AQP5 is dynamically regulated by pathophysiologically relevant stimuli. We hypothesize that cigarette smoke alters the expression and distribution of AQP5 in lung epithelium, contributing to the altered secretions and pathogenesis of chronic bronchitis. To examine this, we propose the following aims: I. Lung epithelial cell lines will be exposed to cigarette smoke and the effects on AQP5 abundance will be examined. Mechanisms underlying these changes will be explored. II. The effects of cigarette smoke condensate on AQP5 subcellular distribution in cultured cells will be assessed by immunofluorescence. AQP5 mutant constructs (phosphorylation sites, ubiquitination sites, truncations) will be used to dissect altered trafficking mechanisms. III. Effects of cigarette smoke on cyclic stretch induced AQP5 expression will be examined. IV. Tracheal epithelial cells from wild type and AQP5 null mice will be exposed to cigarette smoke, and genomic arrays will be performed to assess differential gene expression. We believe these studies will provide novel insights into the potential effects of cigarette smoke on determinants of airway secretions, and provide an important foundation for subsequent examination of the consequences of cigarette smoke exposure on aquaporin biology in nasopharynx and airways of humans.
  
  
  
  
  
• Lars Klareskog, M.D., Ph.D.
  Long-term objectives: To quantify the role of smoking in causing arthritis, to identify mechanisms responsible for this induction and to design methods to prevent or treat smoke-induced arthritis. Specific aims: To use a large case-control study involving recent-onset RA patients as cases, to determine in detail the risk for arthritis development in different groups of individuals exposed to different doses of cigarettes smoke. To use existing well characterized animal models for arthritis to describe molecular pathways that are triggered by smoke exposure and which are involved in the development of arthritis. To use this molecular knowledge obtained on smoking and genetic constitution in animals, to develop new strategies for prevention and treatment against arthritis. Methods: The existing case-control study is comprised of close to 1700 cases of new onset RA and equal number of control individuals, from whom detailed information on the exposure to smoke as well as other potential risk exposures were collected for the time before disease onset. Blood samples enabling genetic analysis are obtained from almost all cases and most controls. In rodent models for arthritis, a genetically defined molecular pathway leading to arthritis has been identified, that makes use of the oxidative burst system. Interference with this oxidative burst has been demonstrated to prevent or cure arthritis. Work plan: 1. To quantify in detail the risk for arthritis development after exposure to defined amounts of smoke with the help of the described large case control study. 2. To define interactions between smoke exposure and genetic all types that determine the function of molecular pathways potentially involved in arthritis induced by smoking, in particular those involved in oxidative burst. 3. To use experimental arthritis models to identify polymorphic genes that determine functions of oxidative burst systems, possibly involved in smoking-induced arthritis. 4. To use the knowledge obtained on genes and molecular mechanisms involving smoking-induced arthritis, to develop new therapies that influence the oxidative burst, and which has in preliminary experiments been shown to alleviate experimental arthritis. Significance: We suggest that the present project will help to develop both preventive and therapeutic measures specifically directed towards smoking-induced arthritis.
  
  
  
  
  
• Yijun Liu, Ph.D.
  The objective of this project is to characterize the brain activity that is associated with serum leptin levels in subjects exposed to environmental tobacco smoke (ETS). It has been found that serum leptin levels in tobacco smokers are significantly higher than those in non-smokers. Recent clinical studies have strongly implicated elevated leptin in cerebrovascular and cardiovascular diseases. Although tobacco smoking induces craving and addiction in smokers, the chronic effects of secondhand smoking on the brain have not been investigated, especially for those effects associated with physiological or endocrinal changes. This is partially due to the lack of suitable human models for directly examining the brain activity in vivo that is responsible for the regulation of those changes, including the blood pressure/heart rate, as well as biochemical signals such as the circulating leptin. We will use both biochemical and neuroimaging methods to determine possible impairment of neural-endocrinal interaction in subjects with exposure to ETS. Specifically, functional MRI (fMRI) will be used to correlate brain activation signals with simultaneous hormonal response measurements during and after food ingestion, focusing on the hypothalamus and its associated neural circuits involved in the regulation of leptin. We hypothesize that the functional regulation of leptin in the brain may be impaired due to secondhand smoking, leading to elevated blood leptin levels, and this impairment may contribute to ETS-related conditions in cerebrovascular and cardiovascular diseases. This hypothesis will be tested by accomplishing following two specific aims. Aim 1: to test the working hypothesis that leptin is associated with central anorexic regulatory pathways localized in the hypothalamus, and this functional regulatory mechanism is impaired in subjects chronically exposed to ETS. Aim 2: to test the working hypothesis that leptin may modulate brain response to smoking-related cues during hunger and sated conditions, and this modulation on the affective (e.g., anxiety-inducing and hedonic) processes may be altered by smoking or secondhand smoking. Furthermore, without the reinforcement effects as in regular smokers in which the ÒhungerÓ signal-associated stress may be attenuated, secondhand smokers may have exaggerated stress-like effects (to be shown by both hormonal and brain changes by fMRI), which may put them in even a higher level of risk associated with cardiac and/or vascular diseases. The proposed project is innovative, and the outcomes of this study will have significant impact on our understanding of how the brain has been chronically affected by smoking or secondhand smoking.
  
  
  
  
  
• James W. Rocco, M.D., Ph.D.
  The goal of this project is to develop an oncolytic adenovirus that replicates based on the status of the Ink4a/Arf locus. Our discovery that p14Arf can suppress adenovirus replication has created the potential to design an adenovirus that only replicates when the p14Arf gene is missing or inactivated. Since this locus is targeted by homozygous deletion in approximately 60% of HNSCC, there is an opportunity to target the majority of head and neck cancer with such a genetically modified oncolytic adenovirus. In our preliminary data, we demonstrate that p14Arf inhibits adenovirus replication at an early step in the virus life cycle, prior to DNA replication. We also show that p14Arf regulates adenovirus replication in a p53-independent manner, which has important therapeutic implications considering the high percentage of HNSCCs that have inactivating mutations in the p53 gene. Further, we confirmed that p14Arf is a potent inhibitor of E2F transactivation, which is essential for adenoviral replication. Finally, using p14Arf specific RNAi, we discovered that endogenous p14Arf modulates the susceptibility of immortalized cells to adenovirus replication, a result consistent with our hypothesis that adenoviral replication is influenced by cellular p14Arf levels. To develop an oncolytic virus as a therapeutic tool we have proposed experiments that will further characterize the molecular mechanisms behind p14Arf regulation of adenoviral replication. As a first step, we will map the specific minimum molecular domain(s) within p14Arf required to regulate adenovirus replication, to further our understanding of the precise mechanism of p14Arf action. Second, we will characterize the interacting domains between p14Arf, E2F and DP1 and determine the relative contribution of p14Arf regulation of E2F transactivation to adenoviral replication. Third, we will characterize the interdependence of the adenoviral E4 gene and p14Arf in adenoviral replication by assessing E4 promoter activity and attempting to rescue p14Arf inhibition of adenoviral replication by expression of the E4(ORF3) and E4(ORF6) genes. Based on these results, we will begin construction of mutant adenoviruses whose replication is dependent upon cellular p14Arf status. Regarding treatment potential, of great significance is that Ink4a/Arf loss occurs early when normal epithelium progresses to cancer and is often seen within the field cancerization surrounding existing lesions. Consequently, an oncolytic virus whose replication depends on p14Arf gene status has the potential to treat premalignant lesions before they progress into cancer, as well as, suppress the development of second primaries in cancer survivors (currently a major source of treatment failure).
  
  
  
  
  
• Charles Rudin, M.D., Ph.D.
  Lung cancer is the most common cause of cancer-related death in both men and women. The large majority of lung cancer cases can be directly or indirectly attributed to tobacco exposure. Aberrant expression of Bcl-2 and/or related apoptotic inhibitors is common in both small cell and non-small cell lung cancers, and results in increased cancer cell resistance to both chemotherapy and radiation. Up to 90% of small cell lung cancers demonstrate expression of Bcl-2. However, our initial clinical studies of an antisense oligonucleotide directed against the bcl-2 mRNA in patients with small cell lung cancer did not markedly alter clinical outcome for these patients. Correlative data from our most recent trial suggests that this may be due to a lack of adequate suppression of the target, Bcl-2, by this antisense molecule in vivo. Recently our laboratory has generated preliminary data demonstrating that newly identified small molecule inhibitors of anti-apoptotic Bcl-2 family members, isolated through a combination of 3-dimensional structural modeling and candidate screening, are highly cytotoxic to lung cancer cell lines, and markedly enhance the efficacy of standard cytotoxic therapy. This proposal seeks to build on these initial data by confirming the mechanism of action of these small molecule inhibitors, by conducting cytotoxicity and chemotherapeutic synergy studies in a panel of lung cancer lines in vitro, and by evaluating single agent and combinatorial antitumor activity in vivo through the use of human lung cancer xenografts in mice. The goal of these experiments ultimately is to promote clinical development of a novel set of cytotoxic agents that function through inhibition of the critical anti-apoptotic pathways upregulated in lung cancer and other malignancies.
  
  
  
  
  
• Barry C. Starcher, Ph.D.
  Cigarette smoking is the most preventable cause of morbidity in the United States and causes more than 3 million annual deaths worldwide. While cigarette smoke is also implicated in the pathogenesis of squamous cell carcinoma of the skin (SCS), little is now known about the pathogenic underlying mechanisms. Prolonged or repeated exposure to solar UV irradiation is another major risk factor for SCS. We therefore infer that development of SCS is promoted by interactions between carcinogens in cigarette smoke and UV irradiation, resulting in oxidative damage to the skin. Cigarette smoke and UV light both increase oxidative stress. Our hypothesis is that cigarette smoke combined with UV exposure causes oxidative and accelerated DNA damage in the skin as well as immunosuppression, which promotes the development of skin squamous cell carcinomas. We address this hypothesis in three interrelated specific aims: AIM 1: To compare the effects of side stream cigarette smoke exposure alone and in combination with UV irradiation on the severity and incidence of skin tumors, dermal matrix changes, and the degree of immunosuppression that occurs in the skin of hairless mice. AIM 2: To determine the amount of oxidative damage that occurs following UV and passive cigarette smoke exposure by assay of mouse urine for 8-oxodG, 8-oxoGua and 8-oxoGuo, and for ethenodA. AIM 3: To determine the ability of selected creams incorporating sun blockers and antioxidants to block skin damage and SCS induced by exposure to cigarette smoke and UV radiation. SKH-1 hairless mice will be exposed to UV irradiation followed by exposure to side stream cigarette smoke. The incidence and severity of tumor development will be documented over 30 weeks. Skin histology and biochemical measurements of collagen and elastin will be conducted at the end of each experiment. Oxidative markers in urine will be serially determined by HPLC-electrospray tandem mass spectrometry. Urine cotinine will be measured as an index of smoke exposure. The ability of creams containing UV blockers and antioxidants to prevent UV and cigarette smoke-induced skin cancer will be tested. The proposed studies will address for the first time mechanisms by which superimposed passive cigarette smoke and UV irradiation cause neoplasia and skin matrix changes.
  
  
  
  
  
• Robert S. Stearman, Ph.D.
  Eicosinoid pathway metabolites are proving to be potent biochemicals with potential therapeutic actions in many diseases. Prostacyclin levels and their relative ratios to each other may be important for inducing inflammatory responses in several pulmonary diseases such as lung cancer and pulmonary hypertension. In particular, the enzyme prostacyclin synthase (PGIS) produces the metabolite PGI2, which is believed to be protective in these pulmonary diseases. PGIS levels are reduced in human lung cancer tissue at both the levels of mRNA and protein. Transgenic mouse models over-expressing PGIS have produced striking results in the prevention of lung cancer after exposure to chemical and smoking carcinogenesis protocols. These findings have led to the rapid approval of a clinical trial for the drug Iloprost, a PGI2 analog, in testing its efficacy in the chemoprevention of human lung cancer in high-risk individuals. In order to determine the mechanism for PGIS down-regulation, we hypothesize that reduced expression from the PGIS promoter is due to epigenetic events that may cause down-regulation of the promoterÕs activity thus leading to disease. We will use appropriate experimental approaches to test three different potential mechanisms for PGIS promoter down-regulation with the goal of ultimately examining laser captured micro-dissected human tissue. The mechanisms being considered are complete loss of one or both alleles of the PGIS gene (LOH), enhanced CpG methylation of the CpG island sites within the PGIS promoter, and changes in the number or sequences of the AP-2 sites that are tandemly repeated proximally to the initiation methionine. LOH can be detected on a cell by cell basis using FISH analysis. Changes in CpG methylation can be determined at the cellular level using methylation specific PCR. Finally, small sequence changes in the PGIS promoter will be characterized by DNA sequence analysis of a number of clones derived from the tissue samples. Initial studies will focus on human NSCLC cell lines to define sufficiently robust and sensitive conditions detecting these events. After verification of the methods, micro-dissected human normal and cancer lung tissues will be assessed for the occurrence and frequency of these events.
  
  
  
  
  
• Yunchao Su, M.D., Ph.D.
  Side-stream cigarette smoke has been implicated as a major risk factor in chronic obstructive pulmonary disease (COPD). Emphysema is the major pathological change in the lungs of COPD patients associated with cigarette smoke exposure. Angiogenesis takes part in the repair process of lung tissue and is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the structural remodeling associated with emphysema. Impairment of angiogenesis plays an essential role in the formation of emphysematous lung. Epidermal growth factor (EGF) is an important mediator released during cigarette smoke-induced airway inflammation and is involved in lung tissue remodeling in COPD. The objective of this study is to test our novel hypothesis that sidestream cigarette smoke extract (SCSE) inhibits EGFÕs angiogenic effect and that this inhibition is caused by decreased calpain activity involving actin cytoskeleton reorganization. To test this hypothesis, we will study the effect of SCSE on EGF-induced increases in calpain activity and protein content and in endothelial angiogenesis in human pulmonary microvascular endothelial cells and will assess whether increases in calpain activity prevent SCSE-induced decreases in EGFÕs angiogenic effect and SCSE-induced alterations in actin cytoskeletal organization. Increases in calpain activity will be achieved by over-expression of subunits of calpain and by gene silencing of calpastatin using antisense and RNA interference technologies. The successful completion of this project will advance our understanding of the regulation of repair of endothelium and tissue in the lungs of patients with exposure to environmental tobacco. Proof of the role of calpain in SCSE-induced inhibition of EGFÕs angiogenic effect may provide a strong rationale for manipulating calpain in the treatment of tobacco smoke-related pulmonary disorders such as emphysema and pulmonary hypertension.
  
  
  
  
  
• Scott Wadler, M.D.
  The Early Lung Cancer Action Project (ELCAP) led by Weill Medical College of Cornell University is an established program to use periodic computed tomographic scanning to screen adult smokers for early, curable lung cancer. The current application is for a subproject within the ELCAP program. The short-term aims are to perfect the methodology for analyzing aspirated clinical samples from early lung lesions, to determine patterns of gene expression in these lesions, and to generate a model to be tested in a future study. The long term aim is to correlate patterns of gene expression identified in this project with clinical outcome in order to improve the management and to reduce the overall mortality from lung cancer in patients with early lung lesions. To date, 17 samples have been analyzed. Ten samples using either the Quiagen or Ambien kits yielded sufficient RNA for analysis; an alternative buffer system was discarded. The final mean cRNA concentrations prior to Affymetrix analysis was 36 ± 21 ucg indicating that sufficient RNA is present for analysis. The present call using the U133A chip was 43 ± 6%, indicating a high degree of informativity. There were 15,000 genes analyzed for each of 10 samples. Heirarchical clustering analysis demonstrated three groups relative to normal lung tissue; all normal tissues clustered. Our plan is to employ microarray analysis of all aspirates of early lung lesions obtained through the ELCAP program using the Affymetrix system with UI33AIB chips, which contain probes for 33,000 human genes. Data will be normalized and filtered, then analyzed by heirarchical clustering software to classify early lung lesions by patterns of gene expresion. Subsets will be correlated with patient demographics, CT findings, cytology and in subsequent studies with clinical outcomes (requirement for surgery, type of surgery, time to recurrence, survival). Subprojects will compare: (a) samples analyzed by laser capture microdissection versus undissected samples and (b) analysis of amplified aspirates versus unamplified surgically resected specimens.
  
  
  
  
  
• Jeffrey S. Wolf, M.D.
  Chronic sinusitis is one of the most common medical problems in the United States. Evidence has shown that it is increasing in incidence and has significant quality of life implications. Although it has been suspected that tobacco smoke exposure is a risk factor and causal agent for sinusitis, the literature regarding this lacks objective evidence and is somewhat contradictory. We have developed an objective strategy to prove our hypothesis that tobacco smoke exposure is a significant contributor to the severity of chronic rhinosinusitis. This study is designed to address two specific aims: Specific aim 1: To demonstrate that there are measurable differences in ciliary structural changes, sinus mucosal cotinine levels, sinus mucosal mitochondrial DNA quantity, patient sinus quality of life measures, and CT radiographic changes among primary smokers (cases) and non-exposed patients (controls). Specific aim 2: To demonstrate objective signs of tobacco smoke exposure and damage in the sinuses of patients in contact with environmental tobacco smoke. This study is a case-control prospective study that enrolls patients with chronic sinusitis that is refractory to medical therapy and who are scheduled for sinus surgery. Prior to surgery, patients will give a detailed tobacco smoke exposure history (both primary and environmental exposures) and complete a validated sinusitis-related quality of life tool. A radiologist will perform a blinded grading of the severity of disease on CT scan. During surgery, biopsies of the maxillary sinus mucosa will be performed. The mucosa will be evaluated for ciliary ultrastructural damage, quantity of mitochondrial DNA, and quantity of the mucosal cotinine. The ciliary damage will be graded by a pathologist using electron microscopy. The quantity of mucosal mitochondrial DNA has been shown to correlate with amount and length of tobacco smoke exposure. The presence of cotinine, a metabolite of nicotine, will be used as a marker of recent tobacco smoke exposure in the sinus mucosal. Multiple variable linear regression analysis will be used to model the dependent variables used to assess chronic sinusitis. By identifying the role of tobacco smoke in the etiology and severity of chronic sinusitis, not only may future strategies for preventing and treating sinusitis be developed, but this will improve program and policy development related to environmental tobacco smoke exposures.
  
  
  
  
  
• Richard J. Wong, M.D.
  Squamous cell carcinomas of the head & neck and lung are highly associated with tobacco exposure. Attenuated, engineered, oncolytic herpes simplex viruses (HSV) are promising therapeutic agents that have significant efficacy in treating a wide variety of malignancies in animal models, including tobacco-related squamous cell carcinoma. These replication-competent, engineered herpes oncolytic viruses have been attenuated for clinical use, and recently completed phase I clinical trials have shown encouraging safety data. Malignant tumors may have varying levels of susceptibility to herpes oncolysis. For successful herpes viral infection to occur, a viral particle must attach to a cellular surface receptor, allowing for fusion of the herpes viral envelope with the cellular plasma membrane. Our goal is to determine if the herpes viral receptor status of squamous cell carcinoma may be used as a determinant of sensitivity to oncolytic HSV therapy. We plan to assess the expression of several herpes viral receptors (HveA, HveB, HveC, 3-OST) in a panel of thirty human head & neck and lung squamous cell carcinoma lines as well as in non-neoplastic fibroblast cell lines. We will use immunofluorescence microscopy, FACS, Western blot, and quantitative RT-PCR to determine the HSV receptor profile for each cell line. Using a replication-competent, attenuated oncolytic HSV (NV1023) we will perform LDH cytotoxicity assays, viral replication assays, and lac Z histochemistry on these cell lines to determine the relative sensitivity of each cell line to viral oncolysis, replication, and infection. We will determine if malignant cell lines have increased expression of herpes viral receptors in comparison to non-neoplastic fibroblast lines. Multivariate statistical methods will then be used to correlate the herpes viral receptor status with sensitivity to viral cytotoxicity (or replication and infection as alternate endpoints). A receiver operating characteristic curve analysis will be performed. We will seek to develop a Òherpes oncolytic prediction modelÓ that may be applied to patient biopsy specimens as a predictor of response to HSV therapy. As oncolytic HSV continue to move closer towards clinical application, issues regarding patient selection remain to be addressed. In our plans for designing a future oncolytic HSV clinical trial, our goal is to use such a model to guide the selection of patients with the most herpes-responsive squamous carcinomas for oncolytic therapy.
  
  
  
  
  
• Michael Mingzhao Xing, M.D., Ph.D.
  Thyroid cancer is the most common endocrine malignancy, with nearly 20,000 new cases per year in the United States, occurring more commonly in women than men (3:1). Several histological subtypes have been identified, each with unique clinicopathological characteristics and presumably with a distinct genetic basis. The most common genetic and epigenetic alterations in thyroid cancer include Ras mutations, RET/PTC rearrangements, PAX8/PPAR translocation, and the BRAF mutation and RASSF1A methylation that we and several other groups recently reported in thyroid cancer. With the exception of childhood radiation exposure causing RET/PTC, no other environmental factors have been associated with these genetic/epigenetic alterations in thyroid cancer. Smoking is known to be associated with certain genetic changes, such as Ras mutations, in other cancers. Furthermore, smoking is strongly associated with nodular goiter and hyperthyroid GravesÕ disease, which have both been associated with increased risk of thyroid cancer. Thyroid cancer in GravesÕ disease is more aggressive. Some of the genetic alterations, such as BRAF mutations and Ras mutations, are similarly associated with a more aggressive phenotype and a poorer prognosis for thyroid cancer. Therefore, we postulate that smoking may cause a higher incidence of certain genetic/epigenetic alterations in thyroid cancer leading to adverse pathological and clinical consequences. One of the major aims of this project is then to investigate whether smoking is associated with a specific genetic/epigenetic alteration in thyroid cancer with predilection to a particular subtype. The common genetic/epigenetic alterations in various subtypes of thyroid cancer will be analyzed with standard molecular biology techniques and correlated with smoking history of the patient. Possible synergic effect of smoking with the genetic/epigenetic change on tumor behavior will also be examined. Another aim is to apply genetic/epigenetic information to thyroid cancer diagnosis. Specifically, the diagnostic accuracy of detecting the cancer-specific BRAF mutation on fine needle aspiration biopsy (FNAB) specimens will be evaluated. The use of a combination of BRAF mutations with other specific genetic markers will also be explored to increase the diagnostic sensitivity. FNAB is widely used but is often limited by indeterminate cytological findings. Use of specific diagnostic genetic markers is expected to amend this deficiency. Smoking patients could particularly benefit from this approach if the genetic markers prove to be associated with smoking as postulated above. An additional aim of this project is to use thyroid tumor cell lines with known status of the activating mutation to test the Raf kinase-specific inhibitor Bay 43-9006. This inhibitor is being proposed for a multi-center clinical trial on thyroid cancer patients incurable with currently available therapies. Such in vitro studies will provide critical pre-clinical support for this trial. Overall, this genetically oriented project will address several critical issues related to genetic epidemiology of smoking, diagnosis, and treatment of thyroid cancer and is expected to have important impact on the practice of thyroid cancer medicine.
  
  
  
  
  
• Cynthia A. Zahnow, Ph.D.
  Background: Passive and active smoking are associated with an increased risk of breast cancer and tobacco carcinogens have been shown to regulate the epidermal growth factor receptor (EGFR) signaling pathway. Our laboratory has recently identified a novel translational regulatory protein known as CUG Binding Protein 1 (CUGBP1) in mammary epithelial cells that is also regulated by EGFR. We have demonstrated that CUGBP 1 controls the translation of CCAAT/Enhancer Binding Protein 3-LIP (C/EBPI3-LIP), a key regulator of mammary gland growth and emerging oncogene in breast cancer. Objectives: We hypothesize that the carcinogens in second hand smoke can stimulate the epidermal growth factor signaling pathway and lead to the activation of CUGBP 1 and subsequent increase in the expression of LIP in mammary epithelial cells. Our objectives are to investigate in both cultured cells and breast cancer biopsies whether tobacco carcinogens can activate CUGBP 1 through stimulation of EGFR signaling. Additionally, we plan to determine which phosphorylated residues are critical for CUGBP1 activity. Thus, we hope to define a new target for second hand smoke in the breast, the emerging oncogenic pathway: EGFR-CUGBP 1-LIP. Specific Aims: (1). To determine whether the tobacco carcinogens associated with particulate matter (PM) in second hand smoke activate EGFR and CUGBP1 in mammary epithelial cells. (2). To determine if CUGBP1 is activated in breast cancer and whether this activation correlates with the smoking status of the patient. (3). To determine whether CUGBP1 activity is controlled by phosphorylation and to map the sites using liquid chromatography tandem mass spectrometry. Research Design: Particulate Matter (PM), collected from second hand smoke (sidestream smoke) generated by smoking machines, will be solubilized and added to cultured, human mammary epithelial cells. Cells will be tested for regulation of EGFR signaling, CUGBP 1 activity and LIP expression. CUGBP 1 activity will also be analyzed in 40 breast cancer biopsies and compared to that in 40 normal breast samples. The identification of CUGBP 1 residues phosphorylated by EGFR signaling or carcinogens associated with second hand smoke will be determined by liquid chromatography nanoelectrospray tandem mass spectrometry. Relevance: We have identified a novel, EGF-activated RNA-binding protein (CUGBP1) in breast epithelial cells that regulates the translation of a transcription factor (LIP) associated with cell growth and mammary tumorigenesis. The identification of phosphorylated residues in CUGBP1 will aid in the design of new therapeutic antibodies or small molecule inhibitors that can recognize and inhibit the activity of CUGBP1. Thus, if successful, our studies in the breast will lead to the identification of new protein targets for the carcinogens found in second hand smoke.
  
  
  
  
  
• JianLiang Zhang, Ph.D.
  Exposure to secondhand smoke is a cause of chronic obstructive pulmonary disease (COPD), which is characterized by lung destruction and loss of alveolar cells. Cigarette smoke-induced programmed cell death or apoptosis of endothelial cells plays a critical role in pathophysiology of COPD. However, the molecular mechanisms of how cigarette smoke induces apoptosis remain unclear. Nitric oxide (NO), a major gas component in cigarette smoke, is known to inhibit mitochondrial cytochrome c oxidase or complex IV, the most NO-sensitive site of the mitochondrial respiratory chain. Persistent inhibition of complex IV can trigger oxidant-related apoptosis. The hypothesis is that cigarette smoke-induced endothelial apoptosis is mediated through the NO-complex IV signaling, which is being tested in cellular models at molecular level by investigating whether cigarette smoke modifies critical residues of complex IV via nitrosylation and nitration, and whether cigarette smoke modulation/ inhibition of complex IV leads to endothelial apoptosis. Molecular cell biological and biochemical approaches as well as a virus-mediated gene delivery system will be used to elucidate the NO-complex IV signaling in cigarette smoke-related lung cell injury and death. This research will add to current knowledge of the possible mechanisms by which cigarette smoke impacts lung and induces diseases.
  
  
  
  
  
• Sergey I. Zharikov, Ph.D.
  Cigarette smoking, including second hand smoking, has been shown to be associated with endothelium-dependent vascular dysfunction mediated through impaired nitric oxide production by endothelial cells. Endothelial cells generate nitric oxide from L-arginine via the catalytic action of endothelial nitric oxide synthase (eNOS). Recent reports indicate that nitric oxide production by endothelial cells is dependent on the availability of extracellular L-arginine suggesting that delivery of extracellular L-arginine, in addition to eNOS activity, may be a regulatory factor in nitric oxide production. The objective of this proposal is to study the effects of cigarette smoke on L-arginine transport and, in turn, nitric oxide production in pulmonary endothelial cells and to investigate possible mechanisms for the action of cigarette smoke on these processes. In our proposed studies, we will use cultured pulmonary artery endothelial cells, which have proved to be a very good model for studying the L-arginine transporter. In pulmonary artery endothelial cells, the delivery of L-arginine is mediated by the cationic amino acid transporter CAT-1. Our preliminary results show that cigarette smoke induces changes in CAT-1 transport activity and that protein kinase C may be involved in these changes. Based on these findings, we hypothesize (1) that cigarette smoke can affect the CAT-1 transporter in endothelial cells via alterations of phosphorylation of this protein by protein kinase C and (2) that cigarette smoke-induced alteration of CAT-1 transport activity can change the delivery of L-arginine into endothelial cells and, in turn, nitric oxide production, thereby modulating endothelial dysfunction in cigarette smokers. In this proposal, we will identify the isoforms of protein kinase C whose activity is changed in endothelial cells after exposure to cigarette smoke. We will detect phosphorylation and transport activity of the CAT-1 transporter in endothelial cells treated with cigarette smoke. Immunoprecipitation experiments will be conducted to prove a possible direct interaction between the CAT-1 transporter and isoforms of protein kinase C affected by cigarette smoke. Finally, we will determine how cigarette smoke-induced alteration of the CAT-1 transporter affects nitric oxide production by lung endothelial cells. The concept that cigarette smoke-induced alterations in L-arginine delivery can contribute to endothelial dysfunction is a novel one and one that has major implications for vascular diseases associated with cigarette smoking. Elucidation of new ways to affect nitric oxide production in endothelial cells through modulation of CAT-1 transport activity will provide new treatment strategies for tobacco-related diseases.
  
  
  
  
  

Center of Excellence

• Varda Rotter, Ph.D.
  The Weizmann Institute of Science in Rehovot, Israel and the Sheba Hospital in Tel Hashomer, Israel seek to establish a Center of Excellence, The Center for Advanced REsearch on Lung Cancer (CARE on Lung Cancer), to support basic and clinical research with a defined focus on lung cancer caused by second-hand and mainstream tobacco smoke. The Center will link world-renown basic cancer and biomedical researchers from the Weizmann Institute, the premier research institute in Israel, and world-class oncologists based at the Sheba Medical Center, a major research_oriented Israeli hospital known for its pioneering approaches to cancer diagnosis and treatment. Scientists and physicians will work as partners to examine research and patient care issues. The Center will support highly interactive, multidisciplinary and multi-institutional research and bring together some of the brightest minds at an outstanding facility in a single country to combat a major disease caused by mainstream and second-hand smoke. This Center of Excellence will enhance research findings related to exposure to second_hand tobacco smoke. The effect of this program on the early diagnosis and clinical care of patients with tobacco related lung cancer will be significantly greater than the sum of the efforts of the individual researchers working separately. Flight attendants will be provided an integral opportunity to be included in future studies. Through the joint efforts of the individual researchers comprising our center we expect to discover more effective approaches toward the prevention, detection and successful treatment of this disease due to mainstream and second-hand smoke. The Center will be directed by Prof. V. Rotter (Director) of the Weizmann Institute and Prof. G. Rechavi (Co_Director) of the Sheba Medical Center, and will include as P1's also Profs. A. Kimchi, Z. Livneh, M. Oren and D. Wallach of the Weizmann Institute, as well as Dr. M. Krupsky of the Sheba Medical Center. The focus of CARE on Lung Cancer will be on developing innovative approaches towards the prevention, early diagnosis and improved treatment of lung cancer, based on the most recent advances in structural and functional analysis of human genes, greatly assisted the human genome project. This is a very ambitious project, aiming at very important goals. Those can be achieved only through the combination of excellence in basic cancer research, and advanced clinical diagnosis and care. A support of a total of 7.5 million dollars (direct cost) over five years is requested from FAMRI in order to enable such combination to bear significant fruit.
  
  
  
  
  

Young Clinical Scientists

• Alexy V. Bazarov, Ph.D.
  In spite of the progress made in recent years, highly metastatic cancers such as breast carcinomas or invasive cancers such as glioblastomas remain extremely difficult to treat and are generally associated with poor prognosis. The standard technique, surgical resection followed by radiation treatment and/or chemotherapy very often results in remaining cancer cells that attain resistance to the treatment. Over the last recent years, a novel and promising concept has emerged to circumvent tumor resistance in which normal host cells within the tumor are targeted to block tumor growth. One such approach is the attempt to block the formation of new blood vessels (angiogenesis) that is required to supply growing tumors with sufficient oxygen and nutrients. The tumor simulates angiogenesis in part through hypoxia-mediated induction of the dimeric transcription factor complex HIF-1. Overexpression of the subunit HIF-1a is associated with increased tumor resistance to therapy and unfavorable prognosis in several cancer types including breast and brain cancer. Based on these findings, an enormous effort has been made to identify inhibitors of angiogenesis and HIF-1a for cancer therapy of which a few have already entered clinical trials. It is the goal of this proposal to inhibit HIF-1a and angiogenesis in established and bulky mammary carcinomas and glioblastoma, which more resembles the clinical situation, and to combine these strategies to improve efficacy and regress end-stage tumors. We will utilize two genetically engineered murine cancer cell lines from transgenic and orthotopic mouse models (MMTV-neu mammary carcinomas and SV40Tag/Hras astrocytoma), and include two human established and well-studied cell lines of mammary carcinomas (MDA-MB-435) and glioblastomas (U251MG). We will first modify these cell lines by transfecting them with a tetracycline inducible Tet-off system to conditionally express the HIF-inhibiting molecules HIF-1a siRNAs. In the presence of doxycycline, expression is shut off whereas when doxycycline is removed, HIF-la siRNAs are expressed. We will then determine how inhibition of HIF-1a influences growth of established and bulky mammary and brain tumors by injecting the mammary carcinoma and glioblastoma cell lines orthotopically into the appropriate microenvironment (mammary fat pad or the brain parenchyma), and then induce HIF siRNA in vivo by removing doxycycline from the drinking water as soon as mice have developed substantial tumor burden. We will further test whether anti- HIF-1a therapy can be improved by combining the treatment with the potent synthetic antiangiogenic molecule SU11248, a receptor tyrosine kinase inhibitor which blocks PDGFR-b, VEGFR, and Kit signaling. We hypothesize that SU1 1248 will reduce the vascular density in the tumors, producing an environment in which the loss of HIF-1a is specifically deleterious for tumor growth leading to substantial tumor regression. It is notable that antiangiogenic molecules also act synergistically with chemotherapy and radiotherapy and that inhibition of HIF-1a is likely to further improve current standard therapies. These studies are therefore of important value to guide clinical trials and to help improve current treatment modalities for cancer.
  
  
  
  
  
• Hetty E. Carraway, M.D.
  Molecular changes in cancer include epigenetic alterations, such as the abnormal methylation of promoter regions of CpG islands. Molecular techniques based on the sensitive detection of abnormal methylation now exist and hold promise as one such molecular method for detection of tumor cells. Breast cancer, like many other malignancies, has recently been shown to have abnormal methylation of many genes, including some tumor suppressor genes. These changes can serve as the basis for a molecular detection strategy. The management of breast cancer patients can benefit from more accurate methods of determining risk of disease recurrence following surgical resection. While the majority of women with histologically negative lymph nodes will be cured by local therapy alone, approximately 25 to 30% of these patients will go on to recur and die of their disease within ten years time of their diagnosis. A molecular means to more accurately predict recurrence in this group would be desirable. We hypothesize that molecular evidence of lymph node involvement exists in some women with histologically negative lymph nodes and it can be detected by methylation-specific PCR of hypermethylated CpG islands. We expect that the presence of such molecular lymph node involvement will predict a higher risk of cancer recurrence following surgical resection with curative intent. The specific aims of this proposal are: 1) To develop tumor specific DNA methylation changes as a molecular staging system for breast cancer. 2) To examine sentinel lymph node biopsies for tumor specific methylation changes than can be predictive of clinical prognosis. The use of tumor specific DNA methylation changes will be studied as a prognostic molecular marker in those patients with node negative breast cancer. We will use a panel of genes, including AP2, BRCA-1, ECAD, ER, FHIT, GST-pi, HIN-1, pl6Ink4a, and RAR-Beta to look for methylation in the sentinel node and corresponding primary tumor. We hypothesize that this gene panel will be sensitive enough to detect molecular evidence of tumor in the histologically negative nodes. We anticipate that the occult presence of aberrantly methylated alleles within histopatholocially negative nodes will identify patients at greatest risk for recurrence. We propose to use prospective sentinel lymph node biopsy technology in an innovative fashion to subdivide the node negative group of patients into groups with differing prognoses based on molecular evidence of methylated alleles representing nodal metastases. This prospective study will include patients who undergo a primary surgical resection for breast cancer including a sentinel node biopsy. This molecular staging strategy may clarify the role of adjuvant therapy for patients with node negative disease who are at highest risk of tumor recurrence. Finally, a molecular marker would help to identify patients at low risk of recurrence and spare them the exposure to toxic adjuvant chemotherapy.
  
  
  
  
  
• Noam A. Cohen, M.D., Ph.D.
  Rhinosinusitis, one of the most common medical complaints in the United States, is responsible for an estimated 13 million yearly physician visits. This disease affects 16% of the US population with an estimated aggregated cost of 6 billion dollars annually. The underlying pathophysiology of rhinosinusitis is stasis of sinonasal secretions leading to bacterial overgrowth and ultimately to infection. In 1991, 88% of the US population was sufficiently exposed to second hand tobacco smoke to elicit a detectable and measurable nicotine metabolite in the blood. Various components of cigarette smoke have been identified as showing great toxicity toward respiratory cilia. That exposure to second hand tobacco smoke contributes significantly to chronic rhinosinusitis is indirectly supported by the fact that children so exposed require revision sinus surgery for persistent or recurrent rhinosinusitis substantially more frequently than those unexposed. This project will investigate the role of second hand tobacco smoke in chronic rhinosinusitis by correlating exposure with the degree of sinus disease as well as sinonasal ciliary beat frequency. Furthermore, we propose to study those second messenger cascades that are altered by second hand tobacco exposure and their contribution to the development of chronic rhinosinusitis. This work has direct implications for elucidating the effects of second hand smoke on cilia elsewhere in the body contributing to understanding the pathophysiology of asthma, as well as diseases of the female reproductive tract including complications of pregnancy and infertility.
  
  
  
  
  
• Christopher D. Coldren, Ph.D.
  Environmental exposure to tobacco smoke is associated with significantly increased risk of lung cancer and other pulmonary diseases, and while active smoking has historically accounted for 85-87% of all new lung cancer cases, the increased risks of lung cancer persist for more than 20 years after smoking cessation. In recent studies, more than 50% of new lung cancers were diagnosed in individuals not currently using tobacco. Therefore, improved success in decreasing lung cancer rates will rely not only on smoking prevention and cessation, but also on effective chemopreventive strategies. Our previous work has demonstrated that the compound prostacyclin (PGI2) provides extraordinary chemoprotection from the development of tumors in a murine model of lung carcinogenesis. These dramatic results have resulted in the initiation of a clinical trial in which patients at high risk for lung cancer will be treated with the prostacyclin analog, Iloprost. This clinical trial is designed to test our hypothesis that the administration of Iloprost will prevent lung cancer in high-risk current and former smokers. As an adjunct to this ongoing clinical trail, we propose to study the expression profiles in circulating peripheral blood mononuclear cells (PBMCs) as surrogate markers of disease. We hypothesize that the gene expression profiles from circulating PBMCs will provide important biomarkers for the development of lung cancer. When combined with advanced methods for data analysis, microarrays have proven to be extremely useful in the classification of histologically indistinct tumor types and in deducing sets of previously unappreciated tumor subtypes responsible for variations in invasiveness and outcome. Our current proposal seeks to capitalize on these developments of technology and technique, and to apply this relatively non-invasive test (array analysis of peripheral blood) to the powerful patient dataset collected in our chemoprevention trial. We have recently completed a similar study of PBMC gene expression markers in Pulmonary Arterial Hypertension, with positive and highly validated results. Furthermore we have preliminary data in COPD patients to suggest that the expression pattern characteristic of circulating PBMCs is modulated in this disease also. This proposal comprises two specific aims: Specific Aim 1: To collect PBMC gene expression data on the upper and lower quartile of Iloprost trial enrollees, as ranked by a risk index to be developed in collaboration with the SPORE group. Blood for these profiles will be collected prior to the administration of the study medication. This data will be used to develop a series of retrospective patient classification schemes based on the clinical and demographic data. Specific Aim 2: To utilize this data, as well as gene expression profiles collected during the follow-up period of the Iloprost trial to create and test hypotheses related to outcome. This aim directly addresses the overall goal of this study-the identification of biomarkers for the early detection of lung cancer. The Iloprost trial is by necessity a prospective study, and therefore the execution of the second aim of this gene expression study must follow the determination of trial intermediate endpoints. It is anticipated that the execution of the first specific aim will provide additional direction for the second aim. Our proposal is directly responsive to the research interests of FAMRI, as it represents a highly novel translational clinical trial addressing the health needs of individuals with past environmental tobacco exposure but no frank disease. Expression profiling of peripheral blood could prove to be an innovative new tool to utilize for detecting biomarkers of disease and response to treatments.
  
  
  
  
  
• Olga Y. Gorlova, Ph.D.
  Passive smoking accounts for approximately 3,000 lung cancer deaths per year in the United States. As with active smokers, there are likely to be genetically determined interindividual differences in susceptibility to tobacco carcinogens in passive smokers. We hypothesize that the lifetime risk of developing lung cancer in environmental tobacco smoke (ETS) exposed individuals is modified by genetic background, dietary factors, and occupational exposures. Our preliminary research indicates that both smokers and non-smokers who exhibit reduced DNA repair capacity have a lifetime lung cancer probability 2 to 5 fold higher than individuals with normal DNA repair capacity. Our aim is to identify epidemiologic (degree of second hand exposure, dietary factors, family history) and genetic factors (with an emphasis on markers of DNA damage and repair) that predict risk of developing lung cancer in never smokers, and also to estimate the absolute lung cancer risk given the degree of ETS exposure, genetic background and epidemiological risk profile. Hypothesis. Lifetime risk of developing lung cancer in environmental tobacco smoke (ETS) exposed individuals is modified by genetic background, dietary factors, and certain chemical and dust exposures. The aims of the proposed study are: Aim 1. To evaluate the effect of various degrees of ETS exposure on lung cancer risk in never smokers, and to identify the factors (epidemiological and genetic) that modify this risk. 1.1. To estimate the risk of lung cancer conferred by different degrees of ETS exposure in never smokers. 1.2. To complete genotyping for polymorphisms for selected genes in the DNA repair pathways and phase II carcinogen detoxifying genes. 1.3. To identify epidemiological and genetic risk factors for lung cancer that modify the effect of ETS exposure in never smokers, including dietary history, family history of cancer, select chemical and dust exposures, genetic polymorphisms, mutagen sensitivity and DNA repair capacity. 1.4. To identify epidemiological and genetic risk factors for lung cancer that are specific for ETS exposed individuals. 1.5. To identify a high-risk group among never smokers using epidemiological (including ETS exposure) and genetic risk factors and to estimate the fraction of never smokers that belong to the high-risk group. Aim 2. To perform exploratory analysis of clinical characteristics of lung cancer (age and stage at diagnosis and tumor histology) for high-risk versus lower-risk never smoker cases, and to compare these characteristics between ETS exposed and not exposed cases. Aim 3. To develop statistical methodology for estimating lifetime risk (probability over a lifetime) of lung cancer for high-risk and lower-risk never smokers, and to apply this method to our case-control data to obtain actual estimates of lung cancer absolute risk given the level of second hand tobacco exposure and underlying genetic susceptibility. This research builds upon an NCI-funded data and specimen repository from an ongoing lung cancer case control study of genetic susceptibility to tobacco carcinogenesis, with 279 never smokers with lung cancer and 324 controls enrolled, matched on age, gender, ethnicity and never smoking status. Detailed epidemiologic data and a comprehensive panel of phenotypic and genotypic markers of susceptibility are available. We will generate more data by genotyping individuals the genetic data on which are missing. We will use genetic and epidemiological data to identify high-risk group among never smokers and clarify the role of ETS in lung cancer risk in never smokers. We will further develop our method of estimating lifetime lung cancer probabilities and obtain the estimates for high risk never smokers.
  
  
  
  
  
• Balazs Halmos, M.D., M.S.
  Recently it has been recognized that cancers are made up of a heterogeneous population of cells only a small proportion of which have the capacity for long-term self-renewal and clonogenic ability. These cells are termed cancer stem cells and have been successfully characterized in leukemias, breast and brain cancers. The identification and characterization of these cells will be critical for not only a better understanding of the biology of the neoplastic process but also in order to design therapies that are targeted at the population of cells that will ultimately determine the outcome of the patientÕs illness. Only normal resident stem cells have the self-renewal capacity to accumulate the number of mutations required for the development of cancer. Therefore, identification of such stem cell populations from normal as well as tobacco-damaged airway epithelium could provide critical clues to the early steps of the tobacco-induced carcinogenic process and lead to the identification of novel markers of high-risk epithelial changes as well as novel targets for lung cancer prevention. In this proposal, we plan to identify and characterize cancer stem cell populations from readily available primary specimens, such as malignant pleural effusions from patients diagnosed with non-small cell lung cancer. We will establish both progenitor cell line as well as NOD/SCID transplant models and will attempt to identify populations of cells with stem cell characteristics based on flow cytometric sorting. We will compare stem cell and non-stem cell populations with the use of transcriptional profiling in order to identify novel stem cell markers and will attempt to identify critical stem cell genes by the use of RNA interference technology. We will establish progenitor cell line models from both normal as well as tobacco-damaged airway epithelial cells. Using these cell lines we will attempt to identify differences in proliferative capacity, multilineage differentiation as well as transcriptional signatures of normal versus tobacco-damaged airway epithelial stem cells. Our studies could provide novel models for the study of lung cancer as well as precursor lesions of lung cancer and could yield novel markers and targets for lung cancer prevention and treatment.
  
  
  
  
  
• Zhaohui Jin, M.D.
  Lung cancer is the leading cause of death in the United States and it demonstrates a strong etiological association with smoking. Nicotine and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are two important components in cigarette smoke. Nicotine can activate the growth-promoting pathways to facilitate the development of lung cancer. 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. NNK is the most potent carcinogen in cigarette smoke and has been implicated as a significant cause of tobacco related tumors. NNK-promoted cellular survival is supposed to be a critical step in NNK-induced cancer development. Although nicotine or NNK 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. Bad is a potent proapoptotic BH3-only member of the Bc12 family that is expressed in both small-cell (SCLC) and non-small_-cell lung cancer (NSCLC) cells. Phosphorylation of Bad results in inactivation of its proapoptotic function. It is possible that nicotine or NNK may induce Bad phosphorylation to abrogate its apoptotic effect leading to increased survival of human lung cancer cells. Our preliminary data show that both nicotine and NNK can activate PKC, ERK1/2 and AKT that are physiologic Bad kinases. Nicotine or NNK potently stimulates phosphorylation of Bad in association with increased cell survival. We hypothesize that the mechanism(s) involved in nicotine or NNK-induced inhibition of apoptosis may occur, at least in part, through phosphorylation of Bad. To critically test this hypothesis, we have identified two specific aims: (1) To determine whether phosphorylation of Bad is required for nicotine or NNK-induced lung cancer survival and chemoresistance. Studies will identify the site(s) of nicotine or NNK-induced Bad phosphorylation in vivo. (2) To determine whether inhibition of nicotine or NNK-induced Bad phosphorylation enhances chemosensitivity in human lung cancer cell lines. 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 fundamental gaps in our knowledge regarding the signaling mechanism by which nicotine or NINK regulates Bad 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 evolve.
  
  
  
  
  
• Sunil S. Karhadkar, MBBS
  In 2003, ~57,000 new cases of urinary bladder cancer will be diagnosed in the US and 12,000 will die of this disease. In men, nearly 50% of these tumors are cigarette-related. Smoke-derived toxins in urine elicit a cycle of mucosal injury and repair linked to carcinogensis in many tissues. We present preliminary data indicating that signaling by Hedgheog (Hh) pathway links bladder injury and repair with bladder cancer. Bladder denudation due to chemical injury is accompanied by a brisk proliferative response that coincides with the expression of the Hh pathway target genes Patched and Gli. Hh pathway target genes are also activated in 4 of 10 bladder cancer lines studied and pathway blockade with the plant-derived Hh antagonist cyclopamine abolishes in vitro growth of these lines. Although cultured cell lines may not accurately reflect actual human tumor biology, ~40% of primary human bladder cancer may also have increased Hh pathway activity, as suggested by cDNA microarray analyses. However, these results were never confirmed. We propose to evaluate the role of Hh pathway activation in bladder cancer. Our specific aims are to I. Estimate the prevalence of Hh pathway activation in human bladder cancer using quantitative RT-PCR for Hh pathway targets, II. Evaluate the efficacy of Hh pathway blockade with cyclopamine in bladder cancer in newly established human bladder cancer xenografts, and III. Characterize the role of Hh signalling in the repair of chemical bladder injury. The proposed research should provide important insights into the pathogenesis of bladder cancer and may indicate a strategy for mechanism-based therapy for this disease.
  
  
  
  
  
• Venkateshwar Keshamouni, Ph.D.
  Lung cancer is the leading cause of cancer death in United States, reaching pandemic proportions with over 150,000 deaths expected each year. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers. The dismal five-year survival rate (10-13%) highlights the need for newer and innovative approaches to treat this disease. Peroxisome proliferator-activated receptor (PPAR)-g is a member of the nuclear hormone receptor super family of ligand-activated transcription factors and functions as a crucial regulator of cellular differentiation. Since cancer cells de-differentiate prior to acquiring the malignant phenotype, inducing differentiation in tumor cells has emerged as an important therapeutic strategy. Consistent with this concept, preliminary studies from our laboratory show that PPAR-g agonists can inhibit the growth of lung cancer cells both in vitro and in vivo. However, the exact role of PPAR-g in carcinogenesis is not known. In the current proposal, we will investigate the role of PPAR-g in the initiation and progression of NSCLC tumors and develop new strategies to target PPAR-g for NSCLC therapy by pursuing the following specific aims: 1) investigate the role of PPAR-g in tumor initiation and progression of lung adenocarcinoma using a conditionally activatable K-ras mouse model; 2) assess the effect of adenoviral_mediated delivery of constitutively active PPAR-g as a potential therapeutic strategy for NSCLC; 3) investigate the potential synergy between PPAR-g ligands and selected chemotherapeutic drugs in NSCLC treatment; and 4) to determine whether PPAR-g gene expression coorrelates with its activity and/or patient prognosis in NSCLC. Collectively, these studies will define the role of PPAR-g in lung tumorogenesis and potentially identify novel therapeutic strategies for this devastating disease.
  
  
  
  
  
• Stephen Y. Lai, M.D., Ph.D.
  Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive malignancy. Despite aggressive surgical and medical interventions, overall survival for HNSCC patients remains relatively poor. Mortality is usually associated with locoregional tumor invasion into vital structures, including the airway. Understanding the molecular mechanisms underlying HNSCC invasion is critical for the development of new strategies to eradicate this disease. Growth factors and cytokines, such as epidermal growth factor (EGF), have been implicated in HNSCC progression and invasion. Although erythropoietin (EPO) was initially characterized as a hematopoietic growth factor, EPO and its receptor (EPOR) have recently been identified in non-hematopoietic cells, including brain and breast. Moreover, EPO and EPOR are expressed by non-hematopoietic human cancers, including breast cancer and neuroblastomas. We have recently identified EPO and EPOR expression in HNSCC cell lines and tissue specimens. The role of EPO and EPOR in human cancers has not been carefully studied and is unknown in HNSCC. We hypothesize that signaling through the EPO/EPOR complex in HNSCC promotes invasion. Preliminary data demonstrates that EPO stimulates HNSCC cell invasion and that the JAK-STAT pathway is activated by EPO stimulation in HNSCC cells. We will determine the role of JAK-STAT signaling in EPO/EPOR-mediated invasion by HNSCC cell lines. A diverse array of inhibitors and signaling intermediate mutants will be employed in analyzing this potential signaling effect. Additionally, the description of EPO and EPOR expression in HNSCC is completely novel. We will analyze HNSCC tissue specimens from our patients for EPO and EPOR expression levels. Our studies will explore potential relationships between EPO/EPOR expression and HNSCC disease stage. We will also examine potential differences in EPO/EPOR expression levels between primary tumors and their metastatic deposits. Additionally, we will examine the pattern of EPO and EPOR expression within HNSCC tissue specimens. The spatial relationship between EPO and EPOR expression within these tissues may suggest potential roles for EPO/EPOR-signaling in HNSCC. Such findings will serve as the basis for exploring possible molecular mechanisms and signaling pathways involved in EPO-mediated HNSCC disease development. The characterization and definition of EPO/EPOR expression and signaling in HNSCC cells will directly impact current patient treatment, as EPO is presently given to HNSCC patients with disease- or treatment-related anemia. Future studies will be directed at other EPO/EPOR-mediated effects in HNSCC, such as proliferation and anti-apoptosis. Understanding the role of the previously unrecognized EPO/EPOR-signaling pathway within HNSCC may lead to the identification of additional targets for future therapeutic interventions.
  
  
  
  
  
• Byron K. Lee, M.D.
  Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia and the most common cause of embolic stroke. AF is also increasing in both incidence and prevalence. The pathophysiology of AF still has not been clearly delineated. Recently, histological data has suggested that inflammation may play a role. C-reactive protein, a marker of inflammation, has been shown to be significantly elevated in patients with AF. Multiple studies also have shown that smoking is associated with elevated C-reactive protein, suggesting that smoking and possibly secondhand smoke may trigger inflammation. This inflammation may in turn affect the atrial tissue and create the substrate for AF. Therefore, we hypothesize that exposure to secondhand smoke increases the risk of developing AF. The Cardiac Electrophysiology Service at UCSF follows approximately 600 patients with pacemakers and 400 patients with implantable cardioverter defibrillators (ICDÕs). The majority of pacemakers and ICDÕs have the ability to record new arrhythmias including AF. We propose a prospective longitudinal cohort study to assess the relationship between secondhand smoke and the development of AF in patients with pacemakers and ICDÕs. Patients without a previous history of AF who are being seen in our clinic for follow-up will be recruited. Patients who smoke, who live with a family member who smokes, or who have work related exposure to smoke will be compared to patients who have no or minimal smoke exposure. Exposure to smoke will be quantitated by means of a validated questionnaire and urinary cotinine measurements. Participants will be followed prospectively for development of AF and other atrial arrhythmias which can be precursors to the AF. The relation of exposure to smoke will be analyzed with adjustment for important baseline characteristics such as age, gender, heart rate, ejection fraction, antiarrhythmic therapy, history of valvular heart disease, history of coronary artery disease, and history of heart failure. We estimate a sample size of 300 patients will result in a greater than 80% power to detect a relative risk of 1.3 between patients exposed to secondhand smoke and those without exposure.
  
  
  
  
  
• Pamela M. Ling, M.D.
  This project addresses the prevention of diseases caused by tobacco smoke by developing a better understanding of how the tobacco industry markets cigarettes to young adults (age 18-24). While the majority of tobacco prevention efforts have concentrated on primary prevention for adolescents, critical transitions from intermittent to regular smoking and addiction often occur during young adulthood. The tobacco industry is well aware of this fact, and has invested years and millions of dollars in sophisticated marketing research on young adults. Tobacco industry researchers have used two strategies that are not routinely used in health research to define market ÒsegmentsÓ (groups of people who respond similarly to marketing messages on the basis of shared needs, characteristics, or behavior). First, segments of young adult smokers were defined by ÒmindsetÓ (general attitudes, activities, goals, aspirations, personality, and brand usage). Second, smokers and nonsmokers have been divided into segments based on their opinions about Òsmoking issuesÓ (secondhand smoke, the social acceptability of smoking, and smoking policies). Using tobacco industry marketing research questionnaires taken from previously secret industry documents, we will collect and analyze new data integrating these two strategies. We will develop a new survey instrument that can identify the different market segments of young adults based on tobacco industry criteria. We will conduct a market segmentation study in a nationally representative sample of young adults to (a) describe attitudes about secondhand smoke in different market segments (b) determine associations between market segment membership and smoking behavior and (c) identify predictors of market segment membership. We will also qualitatively explore responses to tobacco control messages among the members of different market segments. The resulting ability to identify and tailor messages to young adult market segments can be used to improve the efficiency of targeted messaging both for public health campaigns and for patient counseling in clinical settings, which can be expected to make these interventions more effective at blocking the transition from adolescent experimentation to becoming established addicted smokers.
  
  
  
  
  
• Petru Liuba, M.D., Ph.D.
  Previous epidemiological studies have demonstrated a significant dose-dependent increase in cardiovascular morbidity and mortality in adults exposed to environmental tobacco smoke. As atherosclerosis, the underlying disease, begins in childhood under the combined effect of multiple risk factors acting through inflammatory, immune and lipid-related mechanisms, the adverse vascular effects of passive smoking might be initiated already at this early age particularly in children with additional risk factors such as diabetes mellitus. In Sweden, despite several law amendments that ban smoking in public areas, more than 20% of children remain exposed to household smoke. Thus far, an eventual interplay between type 1 diabetes mellitus (DM-1) and ETS has not been investigated. Of note, both DM-1 and ETS increases the susceptibility for recurrent respiratory infections, which have been suggested to contribute to atherosclerosis development. We propose herewith a longitudinal clinical study in which 250 children aged between 1 and 16 years with documented DM-1 (diabetes duration > 1 year), randomly selected from the local Outpatient Clinic, will be followed up for 2 years with yearly controls of both blood status (diabetes status, systemic inflammation, lipid and immune profiles, urine and serum cotinine) and ultrasound-measured markers of early atherosclerosis (endothelium-dependent reactivity of the brachial artery and intima-media thickness of the carotid artery). Sociodemographic information, diet record, medical history (e.g., presence of additional cardiovascular risk factors in parents and patients, prevalence of respiratory, and other acute and chronic infections), and ETS-related information (e.g., number of smokers in the household, number of packs per day smoked by each smoker) will be obtained as well. Possible additive effects of ETS and DM-1 on lipid (e.g., oxidized LDL, total and HDL and LDL cholesterol), inflammatory (e.g., pro-inflammatory cytokines, C-reactive protein, fibrinogen) and immune (e.g., CD-40 and CD-40 ligand) profiles, and on early atherosclerosis (i.e., ultrasound-assessed arterial endothelial dysfunction and intima-media thickening) will be investigated. This study could provide the basis for pediatric interventions aimed to decrease the cardiovascular risk related to the association of diabetes mellitus with ETS.
  
  
  
  
  
• Dana P. McGlothlin, M.D.
  Few epidemics have been as devastating and preventable as that caused by tobacco smoke. Exposure of nonsmoking mothers to environmental tobacco smoke (ETS) has been associated with intrauterine growth retardation, preterm delivery, and low birth weight age infants. The purpose of this proposal is to study the deleterious effects of environmental tobacco exposure on cardiovascular adaptation to pregnancy. Normal pregnancy is associated with significant adaptations in a womanÕs cardiovascular system, including substantial increases in blood volume, cardiac output, and left ventricular (LV) mass. Exposure to tobacco smoke has been associated with an increase in systemic vascular resistance, which may cause an increase in LV afterload and subsequent abnormal hemodynamic and myocardial consequences on maternal cardiovascular adaptation to pregnancy. In this study, echocardiographic and Doppler ultrasound measurements of maternal left ventricular (LV) dimensions and performance will be obtained in two groups of pregnant women during each trimester at weeks 10, 20, and 34 gestation (Group 1 will be nonsmoking pregnant women without exposure to tobacco smoke. Group 2 will be nonsmoking pregnant women exposed to secondhand smoke) to determine if ETS secondhand smoke exposure during pregnancy adversely effects maternal cardiac adaptation. The vascular endothelium modulates vascular smooth muscle tone, and endothelial dysfunction is seen in a variety of pathological conditions, including smoking, atherosclerosis, diabetes, and hypertension. Normal pregnancy is associated with enhanced endothelial function. In this study, we will evaluate endothelial function non-invasively using brachial artery flow-mediated vasodilation (FMD) in the two groups of pregnant women to determine if passive smoking during pregnancy causes endothelial dysfunction. This will be performed at the initial visit, at 34 weeks gestation and 6 weeks post-partum. Exercise can be beneficial to the pregnant woman in the absence of obstetric or medical complications. However, it is important to note that objective data regarding exercise during pregnancy is limited and warrants further research. Pregnancy is associated with several physiologic changes and response to exercise is different in the pregnant state, mainly due to the increase in LV mass and volume. Currently there are no data on the effects of ETS exposure during pregnancy to maternal exercise capacity and cardiac output or to fetal well-being during maternal exercise. The main concern about exercise in pregnancy is that reduced uterine blood flow may cause fetal hypoxia. In this study, we will assess whether secondhand smoke exposure is associated with adverse maternal and fetal cardiovascular responses to exercise in late second trimester during maternal supine bicycle exercise echocardiography. Maternal exercise capacity, blood pressure, heart rate, and echo-Doppler derived hemodynamic responses will be recorded. Fetal echocardiography and Doppler of the umbilical artery will be used to calculate fetal cardiac output and uterine artery flow measurements before, during, and after maternal exercise. Pre- and post-exercise fetal non-stress testing will provide additional information regarding fetal well-being. These results may have implications on clinical recommendations regarding the safety and potential hazards or benefits of exercise during pregnancy in patients exposed second-hand smoke during pregnancy. Pregnancy outcomes will be recorded and transthoracic echocardiography will again be performed 6 weeks post-partum to evaluate potential differences in the reversal of LV structural and hemodynamic changes in patients exposed to tobacco smoke and in older versus younger women. This is the first study to address the potential adverse maternal and fetal cardiovascular responses at rest and during exercise in pregnant women exposed to environmental tobacco smoke.
  
  
  
  
  
• Sarah J. McKinley, M.D.
  Rationale: Emphysema is characterized by long-standing inflammation, increased cell turnover, and the eventual loss of lung parenchyma. Emphysema is caused by exposure to cigarette smoke and becomes increasingly prevalent with age in smokers. Cellular senescence, the cellular manifestation of aging, has been shown to be relevant to the pathogenesis of other chronic inflammatory diseases such as liver cirrhosis. Oxidant stress, known to be caused by cigarette smoke, has been shown to contribute to premature cellular senescence in cell culture. Hypothesis: We hypothesize that cellular senescence of pulmonary parenchymal cells vital in maintaining lung structure produces emphysema in humans. We hypothesize that markers of cellular senescence will be more pronounced in human lung tissue from patients with emphysema as compared to normal control lung tissue. We also hypothesize that markers of cellular senescence will be increased in an existing murine model of emphysema produced by exposure to vascular endothelial receptor 2 blocker, SU5416. We also postulate that in the murine model, as in the human condition, oxidative stress will be important in the development of cellular senescence and hence emphysema. Methods: To test our hypothesis, we propose to evaluate human lung tissue for markers of cellular senescence. Immunohistochemical identification of proteins that inhibit cell cycle progression (pl6TNK4a and p2lCip/Wafl) will be performed. Western blot analysis of nuclear extracts from emphysema and control tissues will be evaluated for pl6TNK4a and p2lCip/Wafl. Enzyme activity of the senescence-specific marker beta-galactosidase in samples will be determined. Telomere length will be assessed using fluorescent in situ hybridization (FISH) and Southern blot analysis. Similar methods will also be used to analyze tissues from an existing mouse model for emphysema (VEGF receptor blockade with Su5416) to establish the relevance of this model to the human disease and to evaluate possible treatments for senescence-induced emphysema.
  
  
  
  
  
• Richard J. Rogers, M.D., Ph.D.
  Cigarette smoking and exposure to environmental tobacco smoke (ETS) are enormous societal problems because of the increased incidence of cardiovascular disease in the population. Nicotine, a potent alkaloid component of tobacco, is quickly absorbed through the lungs and affects the endothelial function of blood vessels in multiple organs. Studies have shown that even low serum nicotine concentrations increase the oxidative stress of the organism by increasing the release of free radicals and serum inflammatory mediators. A crucial anti-oxidant within all aerobic organisms is the mitochondrial enzyme, manganese superoxide dismutase (MnSOD). Fortunately under conditions of oxidant stress, the manganese superoxide dismutase gene responds to cellular inflammatory signals by increasing the MnSOD expression. However, we have found that nicotine at low concentrations and short duration of exposure abolishes this inducible cytoprotective response of the MnSOD gene at both the mRNA and protein levels. This effect of nicotine on MnSOD expression, particularly during oxidant stress, may be of considerable importance in the etiology of cardiovascular disease, stroke and cancer seen in smokers and individuals exposed to ETS. Our global hypothesis states that nicotine alters the transcription of basal and inflammatory mediator-inducible expression of MnSOD. First, we will show that nicotine interferes with new transcription of the MnSOD gene by utilizing nuclear run-ons from isolated human endothelial cell nuclei. Second, we also intend to demonstrate that the phenomenon occurs in human endothelial cells at both the mRNA (transcriptional) level using steady-state northern analysis and the protein (translation) level utilizing western analysis. Our next hypothesis is that nicotine alters basal and inducible MnSOD gene expression through a nicotinic acetylcholine receptor-mediated pathway. First, we will utilize a variety of nicotinic acetylcholine receptor antagonists to block the effect of nicotine on human endothelial cells and assess the results on transcription and translation by northern and western analysis, respectively. Second, by utilizing human embryonic kidney (HEK) cells stably expressing different nicotinic acetylcholine receptor subtypes, the effect of nicotine on MnSOD expression can be assessed at both mRNA and proteins levels. HEK cells do not normally express nicotinic acetylcholine receptors and nicotine does not have an effect on MnSOD in wild-type HEK cells. Our third hypothesis is that nicotine affects MnSOD expression by altering important protein-DNA interactions on the regulatory regions of the MnSOD gene. First, utilizing a plasmid reporter gene system, human growth hormone (hGH), in conjunction with portions of the human MnSOD promoter region and transiently transfecting human endothelial cells with these constructs and subsequently exposing to nicotine, we will demarcate important regions of the MnSOD promoter responsible for the alterations in basal transcription caused by nicotine. Second, by incorporating the human MnSOD enhancer into these human MnSOD promoter region hGH constructs and transiently transfecting human endothelial cells subsequently exposed to nicotine, inflammatory mediators or both, we will further delineate the role nicotine plays in altering the inflammatory mediator-inducible expression of MnSOD. Third, to assess actual protein-DNA interactions within segments of the MnSOD promoter and enhancer, we will perform electrophoretic mobility gel shift assays with isolated nuclear proteins from endothelial cells which are unexposed, exposed to nicotine, or exposed to nicotine and inflammatory mediators.
  
  
  
  
  
• Guilio R. Romeo, M.D.
  Cardiovascular disease (CVD) represents the main cause of morbidity and mortality in developed countries. Major risk factors for CVD, including smoking, are often associated with CVD and are believed to play a role in atherosclerosis. In fact, smoking is the strongest independent predictor of premature coronary artery disease. The molecular bases underlying atherosclerotic lesion formation are not fully understood, especially at the initial stages of the disease. The lack of such information impairs our ability to fully prevent CVD as well as devise adjunct therapeutic interventions. We have previously identified Profilin-1 as a new candidate contributor to atherogenesis. Profilin expression was dramatically increased in both human and experimental atherosclerotic lesions when compared to non-diseased vessels, and was upregulated by LDL and oxysterols in primary cultures of rat aortic endothelial cells. Also, profilin overexpression mediated endothelial dysfunction, which possibly precedes and participates to atherosclerosis. Thus, our hypothesis is that profilin plays a role in the development and/or progression of the atherosclerotic plaque. To test this hypothesis, we propose the following Specific Aims: To test the effects of profilin-1 (pfn) deficiency on the onset and progression of atherosclerotic lesions in the apoE-null mouse model of atherosclerosis. To this end, we will establish a colony of pfn +1- /apoE mice (profilin homozygous knockout are not viable) and study number, and complexity of their lesions in comparison with apoE' mice at different time points. To elucidate the pathway whereby LDL/oxysterols elevate profilin levels. The transcription factor(s) responsible for the LDL/oxysterols-dependent regulation in profilin will be identified. The findings collected in these experiments will address the role of profilin in atherogenesis and its regulation by LDL/oxysterols. The long-term goal of this project is to identify pathogenetic targets (e.g. transcription factors regulating profilin increase) that can be translated into new forms of interventions for CVD. Ideally, this knowledge would also provide candidate markers for the early stages of the disease, which are often silent.
  
  
  
  
  
• Catherine M. Shachaf, Ph.D.
  Tobacco smoke contains some of the most potent carcinogens that have been identified. These carcinogens have been shown to induce genetic alterations that frequently culminate in many types of human neoplasia, in particular, lung cancer. Over 10% of Americans that smoke will eventually acquire lung cancer and only 14% of these individuals will live longer than 5 years. To date, limited progress has been made in the treatment of lung cancer, suggesting that new therapeutic approaches are essential. The majority of tobacco-related lung cancers that exhibit over-expression of the MYC proto-oncogene develop a very malignant form of lung cancer suggesting that this oncogene plays a critical role in the pathogenesis of tobacco-related lung cancers. Previously, we have obtained evidence suggesting that MYC or genes regulated by MYC may be a good target for the treatment of cancer and we hypothesize that inactivation of MYC may be effective in the treatment of tobacco-related lung cancer. To address this possibility, I will use a novel tetracycline regulated mouse model developed in Dr. Felshers laboratory, in collaboration with Dr. Whitsett, to generate transgenic mice that conditionally regulate the expression of MYC in the lung. By treating mice with tetracycline the expression of MYC can be overexpressed or inactivated specifically in the lung. I reasoned that in this manner I could mimic the ability of tobacco to induce carcinogenesis of the lung and test if upon MYC inactivation tumors exhibited tumor regression. I have obtained preliminary evidence suggesting that in our tobacco-related lung cancer mouse model, the induction of MYC overexpression indeed induces lung cancer. Moreover, we found that upon MYC inactivation tumors exhibited a significant decrease in the tumor burden. Therefore identifying key gene regulators involved in the process of lung tumor regression will be important in developing novel therapies for lung cancer. Now, I propose to use this new model system for tobacco-related lung cancer as a pre-clinical model to investigate several questions that will evaluate the effectiveness of MYC inactivation for the treatment of tobacco-related lung cancer. First, I will determine the mechanism by which the tumors regress. I will determine if MYC inactivation induces tumor regression by proliferation arrest, apoptosis and differentiation. Second, I will study changes in gene expression using cDNA microarray analysis and quantative RT-PCR to attribute gen