FAMRI 2002-2003 Funding Cycle Awardees

Clinical Innovators

• Theodore P. Abraham, M.D. 
  Population-based studies have unequivocally demonstrated the association of passive smoking with coronary artery disease and endothelial dysfunction. It is likely that the vascular and cardiac effects of passive smoking extend well beyond those currently documented. In particular, it is unclear whether passive smoking is associated with hypertension. It is also unknown whether passive smoking is associated with other abnormalities such as vascular stiffness or impaired ventricular relaxation. If passive smoking were indeed associated with hypertension, vascular stiffening and impaired ventricular relaxation, there would be tremendous health care implications. This proposal will use standard and state of the art, well-validated, quantitative, non-invasive echocardiographic techniques to study the influence of passive smoking on peripheral vascular and central cardiac physiology. We hypothesize that passive smokers will have higher systolic blood pressure, lower vascular compliance and impaired ventricular relaxation, as compared to non smokers. We will measure ambulatory 24-hour blood pressure, vascular compliance (to evaluate vascular stiffness) and assess regional and global ventricular relaxation. The study group will consist of normal, healthy, non-smoking volunteers with no co-morbidities and not on any medications. We will compare passive smokers to non-smokers. We will also, in a limited fashion, assess whether cessation of passive smoking positively influences peripheral or ventricular physiology, by comparing passive smokers with previous passive smokers. This proposal builds on the experience of the applicant in clinical trials, and the applicant's expertise in echocardiography, specifically, novel, well-validated, quantitative, non-invasive ultrasound techniques that are sensitive to subtle changes in vascular and cardiac mechanics. The research will be conducted at an institution with a strong tradition, and existing infrastructure for clinical research, supplemented by state of the art equipment and trained personnel. The results of this study may influence societal and biomedical strategies concerned with passive smoking. We plan to use the results of this study to launch a larger and longer term study to study the long term biological affects, and the affects of cessation of passive smoking on the cardiovascular system.
  
  
  
  
  
• Douglas W. Ball, M.D. 
  Non-small cell lung cancer (NSCLC), the commonest form of human lung cancer, is overwhelmingly associated with tobacco smoke exposure. Current treatment of NSCLC is hampered by low response rates and frequent relapse. A promising target for experimental therapy of NSCLC is to interrupt growth factor signaling pathways, particularly downstream of epidermal growth factor (EGF). Our laboratory recently has demonstrated novel interactions between the ras-raf-MAP kinase pathway (downstream of EGF and related growth factors) and the developmental regulator, Notch. The Notch pathway is a versatile signaling mechanism with critical roles in stem cell and cancer biology. In normal lung development, the Notch pathway is active in proliferating airway epithelia. Similarly, we find evidence for Notch pathway activation in a majority of NSCLC cell lines, but not in neuroendocrine cancers such as SCLC. We have recently shown that over-expression of activated Notch l markedly induces activation of MAP kinase, implying that Notch and growth factor receptor-ras-raf pathways may interact in lung cancer. Our hypothesis is that Notch signaling in NSCLC promotes growth and survival, through potentiation of growth factor receptor signaling including the EGF receptor (EGFR). Furthermore, we predict that inhibition of Notch signaling, using small molecule inhibitors, may be effective in blocking growth factor actions, especially in combination with inhibitors of EGF signaling. In the proposed studies, we will explore the scope of Notch pathway activation in human NSCLC, using immunohistochemistry in tumor specimens. We will then characterize mechanisms underlying the observed interaction of the Notch and ras-raf-MAP kinase pathways in cultured NSCLC. Finally, we will explore the efficacy of Notch signaling inhibitors in cultured NSCLC, especially where EGFR signaling is reduced via growth factor depletion or receptor inhibition. If these hypotheses are validated, our work will provide important proof-of-principal for intensive efforts to incorporate anti-Notch strategies in combination with other NSCLC therapeutics.
  
  
  
  
  
• Teresa De Marco, M.D. 
  Pulmonary hypertension (PH) is a disorder defined by persistent elevation of mean pulmonary artery pressure >25 mmHg and can lead to progressive right heart failure (RHF) and premature death. PH is classified into pulmonary arterial hypertension (PAH), pulmonary venous hypertension, and pulmonary hypertension due to respiratory diseases, thromboembolic diseases, or diseases involving the pulmonary vasculature. Chronic obstructive pulmonary disease and left cardiac failure account for most cases of pulmonary hypertension in the USA. Processes that contribute to the development of PH include vasoconstriction, vascular remodeling, and thrombosis in situ. Central to the pathogenesis of PH is dysfunction of the vascular endothelium along with platelet and coagulation overactivity. Previous studies have shown that smoking is associated with pulmonary vascular remodeling and passive or active smoking during pregnancy is a risk factor for persistent PH of the newborn. In addition, there are a multitude of studies showing that secondhand smoke (SHS) is detrimental to endothelial and platelet function, oxygen delivery and processing, and exercise capacity in other cardiovascular diseases. We hypothesize that exposure to SHS will abrogate exercise capacity benefits of drugs such as bosentan and epoprostenol in patients with advanced PAH. Furthermore, SHS exposure will increase clinical adverse events in this population. We propose a 1-year longitudinal, prospective cohort pilot study in patients with PAH (as the prototype of PH) which will form the basis for future studies in PH associated with lung disease and left ventricular failure. PAH patients recruited from the PH clinic at the University of California, San Francisco who have exposure (utilizing a validated questionnaire) to SHS will be compared to PAH patients who are not exposed to SHS. The following parameters will be tracked at 3 month intervals: 6-min walk distance, Borg dyspnea index, WHO Functional Class, and clinical adverse events (hospitalization, lung transplantation, stopping previously effective medications, and death). A sample size of 100 will provide 8o% power to detect &Mac179; 75 meter difference in 6- minute walking distance (the primary endpoint) with an alpha of 0.05.
  
  
  
  
  
• Kirsten E. Fleischmann, M.D., MPH 
  Congestive heart failure is a common clinical syndrome, affecting over 2 million people in the United States alone and resulting in extensive morbidity and increased mortality. Recent analyses suggest that smoking is associated with an over 40% increase in mortality over never smokers in patients with congestive heart failure after adjustment for important clinical factors. Moreover, exposure to secondhand smoke has been associated with an increased risk of heart disease and cardiovascular death, with relative risks of 1.3-1.7 depending on the outcome and cohort studied. We hypothesize that exposure to secondhand smoke is also associated with poorer outcomes in patients with congestive heart failure. We will test this hypothesis with a longitudinal cohort study to assess the relationship of exposure to secondhand smoke to death, nonfatal myocardial infarction, and rehospitalization for heart failure. Participants will be recruited from the Heart Failure and Cardiology Clinics at the University of California, San Francisco. Patients with a family member/housemate who smokes or with work-related exposure to smoke will be compared with patients without domestic or work-related exposure to passive smoke. Exposure to secondhand smoke will be quantitated by means of validated questionnaires completed by the study participants and urinary cotinine measurements. Current smokers will be excluded from this study. Participants will be followed prospectively over the course of the grant for outcomes of death, nonfatal myocardial infarction, and recurrent hospitalizations for congestive heart failure. The relation of exposure to secondhand smoke to outcome will be analyzed with adjustment for important baseline characteristics such as age, gender, blood pressure and heart rate at enrollment, previous MI, revascularization history, history of hypertension, smoking history, etiology of heart failure, NYHA class, and ejection fraction. We estimate a sample size of 300 patients will result in greater than 8o% power to detect a relative risk of 1.3 between patients exposed to secondhand smoke and those without exposure.
  
  
  
  
  
• Alexey Fomenkov, Ph.D. 
  Primary and second-hand smoking is one of the major risk factors for head and neck squamous cell carcinomas (HNSCC). The incidence of HNSCC is greatly increased from exposure of the upper aerodigestive tract to environmental tobacco smoking. The epidermal growth factor receptor (EGFR) gene is amplified/overexpressed/activated in the majority of epithelial malignancies including HINSCC, and associated with smoking. Our central hypothesis is that identification of the EGFR downstream signaling intermediates and responsive genes would help to design novel therapeutic targets for HNSCC.
  
  The specific aims are: (1) to determine the biological effects of primary and second-hand smoking on EGFR signaling pathways in HNSCC; (2) to identify the EGFR responsive gene targets in HNSCC from patients with primary and second-hand smoking by chip DNA microarray approach; (3) to determine the effect of EGFR inhibitors on EGFR downstream signaling intermediates and responsive gene targets in HNSCC. Patients with HNSCC and various environmental smoking history will tested in these studies for phosphorylated EGFR and its downstream signaling intermediates. The Affymetrix DNA chip array will be applied to compare gene profiling from smokers, second-hand smokers and non-smokers.
  
  Preliminary studies showed that small molecules-inhibitors of EGFR signaling pathways (OS1-774 or ZD-1839) modulate activation of the Ras/Erk/MAPK pathway, but fail to inhibit PI3K/Akt pathway in HNSCC lines. P63 expression activated by EGF in HNSCC is modulated by ZD- 1839, which implicates EGFR signaling in regulation of p63 expression. This translational project provides an opportunity to extend findings from a preclinical models and pilot observations in HNSCC patients showing the effects of OSI-774 on the EGFR activation and signaling through the Ras/ErkJMAPK and PI3K/Akt pathways. The advantage of testing a population of HNSCC patients with smoking history (primary and second-hand) eligible from our clinical trials is that they will be treatment-naive and have readily accessible tumor for serial biopsies. The data resulting from these studies will assist in finding of novel targets in the EGFR signaling pathway to generate the innovative therapy for HNSCC potentially based on smoking history.
  
  
  
  
  
• Ephraim J. Fuchs, M.D. 
  Population-based studies estimate that 24-30% of the 30,000 cases of kidney cancer diagnosed each year in America are attributable to smoking. A significant number of these patients have metastatic disease at the time of the presentation, and cytotoxic chemotherapy drugs are largely ineffective. Dramatic tumor responses have been seen in some patients treated with interleukin-2 or interferon alpha, suggesting that this disease may be amenable to immunotherapeutic approaches. Recent evidence in the clinic suggests that non-myeloablative allogeneic stem cell transplantation, or NST, is associated with an immunologically mediated "graft-versus-tumor" effect that can mediate the regression of metastatic RCC (mRCC). Unfortunately, NST is complicated by the frequent occurrence of graft-versus-host disease (GVHD), an indiscriminate attack on host tissues by donor T cells, and fewer than 10% of patients undergoing NST for in RCC achieve complete responses of their disease. We have recently demonstrated in the laboratory that autologous tumor vaccines, administered after NST, augment the antitumor effect of allogeneic T cells without exacerbating GVHD. Autologous tumor-derived heat shock proteins (HSPs) also induce therapeutic anti-tumor immunity, because HSPs bind tumor-specific peptides and deliver them, via HSP receptors on immunogenic antigen-presenting cells, for presentation to tumor-specific T cells. These pre-clinical data form the rationale for clinical trials of tumor-derived HSP vaccines after NST for mRCC. The primary objective of the clinical trial, and therefore of this proposal, is to determine the safety and anti-tumor efficacy of autologous tumor-derived HSP vaccines when administered together with donor lymphocyte infusions (DLI) to treat mRCC after HLA-identical NST. The secondary objective of the trial is to characterize the patients' immune response to autologous tumor cells by delayed type hypersensitivity (DTH) testing. Eligible patients with mRCC will undergo removal of the affected kidney, which will serve as the source of tumor-derived HSPs. Following recovery from surgery, patients will receive a transplant of HLA-identical sibling marrow in the context of conditioning with fludarabine, 200 cGy total body irradiation, and post-transplantation cyclophosphamide. Following discontinuation of post-transplantation immunosuppression, patients without GVHD will receive 107 donor CD3+T cells/kg and four HSP vaccinations on a weekly basis. Clinical endpoints include overall and progression-free survival, transplant-related mortality, and incidence and severity of GVHD. Biological endpoints include lymphocyte reconstitution, donor chimerism, and DTH responses to tumor cells as measured before transplant, two weeks after the last vaccine, and at the time of clinical response.
  
  
  
  
  
• Daniel L. Hamilos, M.D. 
  Chronic sinusitis affects 12% of the US population and is even more prevalent among active cigarette smokers. Many nonsmoking flight attendants are also affected by chronic sinusitis. This has prompted the question of whether there is an increased prevalence of chronic sinusitis in individuals with secondhand cigarette smoke (SS) exposure. In Aim #1, we will test the hypothesis that in a randomly selected population of individuals with SS exposure, the prevalence of chronic sinusitis will be greater in subjects with the greater exposure. Toward this end, we will conduct a survey of 2000 randomly selected adults in the St. Louis area with SS exposure and determine the extent of SS exposure and presence or absence of chronic sinusitis. The correlation coefficient between SS exposure level (e.g. 1-5, 6-10, 11-20, 21-40 and > 40 cigarettes/day) and the prevalence of chronic sinusitis at each level will be computed, and we will test the null hypothesis that this coefficient is equal to zero. In Aim #2, we will test the hypothesis that extent of SS exposure and serum cotinine levels will be greater in SS exposed individuals with chronic sinusitis than those without it. We will also test the hypothesis that the extent of SS exposure correlates with levels of inflammation or expression of certain mucin proteins. To accomplish Aim #2, we will recruit two cohorts (N=50 per cohort) from the population studied in Aim #1, namely those with and those without chronic sinusitis. Participants in each cohort will undergo nasal lavage and middle turbinate biopsies for quantification of inflammatory cells, cytokines and mucins, including MUC 2, MUC 5AC and MUC 5B. Correlation coefficients between cotinine levels and various inflammatory markers will be computed for the entire group of 100 subjects, and we will test the null hypothesis that these coefficients are equal to zero. To determine whether any association between cotinine levels and sinusitis is independent of inflammatory markers, multiple logistic regression will determine whether cotinine is a significant predictor after adjusting for age, gender and the inflammatory markers, where the markers included in the latter model are those that are found to be significant using univariate analysis. We hope to identify characteristic features of inflammation and mucin expression in SS exposed individuals with chronic sinusitis. A long-term goal is to apply knowledge from this grant to improved diagnosis, treatment and prevention of chronic sinusitis in a FAMRI-sponsored Center of Excellence.
  
  
  
  
  
• Lori Kornberg, B.S., Ph.D. 
  Head and neck cancers (SCCHN) are primarily associated with long-term exposure to tobacco. Each year 78,000 people in the United States will be diagnosed with SCCHN and 17,500 will die from the disease. This represents 4% of all cancer deaths annually. SCCHN was formerly a predominantly male disease, but as more woman become exposed to tobacco products the disease rate is moving towards sexual equality.
  
  Focal adhesion kinase (FAK) is a tyrosine kinase that mediates intracellular signals generated by the integrin family of adhesion receptors. We have shown that FAK and phosphoFAK are overexpressed in oral and laryngeal cancers. We also generated a transformed epithelial cell line (293) that can be induced to overexpress the FAK inhibitor, FRNK. Overexpression of FRNK led to a decrease in the adhesion-mediated phosphorylation of FAK on tyrosine 397 and a decrease in adhesion-induced phosphorylation of the growth-promoting kinases, Erk 1/2. These cells had reduced migration on ECM proteins. FRNK overexpression did not induce apoptosis, but these cells were more sensitive to the cytotoxic effect of 5-fluorouracil. Based on this data, we hypothesize that inhibition of FAK activity can be used to treat SCCHN. Furthermore, we propose that combination therapies involving FRNK, p53, and cytotoxic drugs will be extremely effective in treating SCCHN. In order to test this hypothesis, the applicant proposes to: 1) Generate an adenoviral vector that can be used to overexpress FRNK 2) Determine the effect of adenoviral-mediated overexpression of both FRNK and wild-type p53 on the behavior of SCCHN cells in vitro. If this work is successful, it would have a very high impact on the treatment of SCCHN.
  
  
  
  
  
• David J. Lee, Ph.D. 
  It has been 16 years since publication of the landmark Surgeon General's Report that linked secondhand smoke exposure to an increased risk of otitis media in children. Since its release three other major reviews have confirmed this association in infants and young children. However, there have been no published studies examining this association in adolescents and relatively few studies completed in younger school-age children. Furthermore, most of the studies included in these reviews are subject to methodological limitations which include absent or limited clinical assessment of middle ear function and/or reliance upon self-reported or parent reported exposure to secondhand smoke. The use of biomarkers of exposure to secondhand smoke is essential since study participants often underestimate their tobacco smoke exposures. This study will overcome these limitations by documenting the association between middle ear dysfunction and secondhand smoke exposure confirmed by serum cotinine levels using data obtained from over 3,100 participants of the National Health and Nutrition Examination Survey III (NHANES III). The NHANES III, which is designed to be representative of the US population, is the only national survey which has collected both cotinine data and has administered clinical measures of middle ear function in participants 4-19 years of age. This study will significantly extend our knowledge of the adverse otologic effects of secondhand smoke exposure in school-age children and adolescents.
  
  
  
  
  
• Pierre P. Massion, M.D. 
  Lung cancer is the number one cause of cancer-related deaths in the United States. The fight against this disease is greatly compromised by a lack of successful early detection strategies. Our goal is to develop and to validate new molecular targets for the early detection of lung cancer. We propose to identify molecular abnormalities in invasive and preinvasive lung cancers using two new technologies: array comparative genomic hybridization (aCGH) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We will validate the findings by determining the frequency of these abnormalities in tissue microarrays (TMA) and by determining the clinical associations in our linked database. Our specific aims are: 1. Identification of amplified and deleted genomic regions in invasive squamous, adeno-, large- and small cell lung neoplasia using aCGH. We will extract genomic DNA from archival tissues after microdissection, and we will use gene-specific aCGH to identify areas of common genomic amplification or deletion found in lung cancer. The prevalence of those abnormalities will be assessed by using tissue fluorescence in situ hybridization (FISH) in aim 3. Priority will be given to genes that are frequently and highly amplified in each subtype. 2. Use of tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to identify potential targets in preinvasive lung neoplasia. Using our fluorescence bronchoscope we will also obtain normal, preinvasive and invasive cancers from bronchial biopsies of patients undergoing bronchoscopy for the suspicion of lung cancer. Using MALDI-MS, we will analyze the protein expression profile of surgically resected human lung cancers and corresponding normal lung tissues. We will identify profiles of proteins expressed in both preinvasive and invasive lesions and validate the level of expression of a selection of proteins most prevalent by immunohistochemistry. 3. Clinical validation of candidate genes/proteins associated with lung cancer progression. Immunohistochemical staining will be performed for best candidates on our 280 patients TMA that includes tumors studies in aim 1 and will be correlated with clinical data elements from our lung cancer SPORE database. Using this strategy we will begin to assemble a molecular model of lung cancer progression.
  
  
  
  
  
• Ramin Mirhashemi, M.D. 
  There are considerable evidence that invasive cervical cancer arise from intraepithelial neoplasia (CIN) and that human papillomavirus (HPV) is involved in their initiation, although HPV infection alone is clearly insufficient to fully explain cervical carcinogenesis. Research priority has been to identify additional factors that may contribute to cervical cancer initiation or progression. Cigarette smoking has received considerable attention with many studies demonstrating a modest but significant increase in relative risk associated with this exposure. The biologic plausibility of this association is strengthened by the finding that the cervical mucosa of women who smoke contains measurable amounts of the potent tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To enhance our understanding of the role of environmental tobacco smoke (ETS) in cervical disease, we are proposing to study the underlying genetic susceptibility to aryl amine and derivative compounds present in cigarette smoke. We propose a genomics and population based design to study the role of xenobiotic metabolism genes in human cervical cancer susceptibility. Because cervical cancers undoubtedly conform to the paradigm of multistep, multigenic carcinogenesis, our hypothesis is that cervical cancer susceptibility is a function of a differential bioactivation of environmental carcinogens in the population and that women with cervical cancer or pre-cancers share certain xenobiotic metabolism gene polymorphisms not present in healthy women We will recruit tobacco exposed and non-exposed women with normal cervical tissue CIN or invasive cervical cancer. A panel of about 750 confirmed SNPs in 32 xenobiotic metabolism genes will be tested in six groups of twenty patients each (women with normal cervical tissue versus CIN versus invasive cervical cancer in smokers and non-smokers). Given the indication in literature that smoking is an independent risk factor for cervical cancer, this study may shed some light into the mechanism of such a risk at the molecular genetic level.
  
  
  
  
  
• Jan S. Moreb, M.D. 
  Our overall objective is to study the role of aldehyde dehydrogenase Class-1 and Class-3 (ALDH-l and ALDH-3, respectively) in chemotherapeutic drug resistance and pathogenesis of lung cancer, a disease causally associated with environmental tobacco smoke (ETS). NSCLC cell lines were found to have the highest constitutively expressed levels of ALDR-1 and/or ALDH-3. These enzymes are involved in the inactivation of active metabolites of cyclophosphamide (CP), a widely used anti-cancer therapy, as well as in the metabolism of acetaldehyde and other carcinogens that are byproducts of ETS. Our hypothesis is that high ALDH-1 and ALDH-3 levels are related to the evolution of the malignant process and the exposure to carcinogens, either directly or indirectly through mediators released during damage caused by carcinogens as well as the activation of new genes (oncogenes, transcription factors, etc.) as a result of DNA mutations, and that downregulation of one or both enzymes will result in increased sensitivity of lung cancer cells to CP and its active derivatives. Two specific aims have been identified: Aim I: To establish the relationship between ALDH-l and ALDH-3 and the pathogenesis of lung cancer. We will compare the level of expression of these enzymes in normal versus malignant lung epithelial cells: We will use established cell lines for normal lung epithelium and lung cancer, as well as samples obtained from patient lung tissue with or without lung cancer. The enzymes will be quantified mainly by immunohistochemical staining, but also spectrophotometric enzyme activity assay, Western and Northern blots as needed. These studies will determine the association between ALDH-1/ALDH-3 expression and the morphologic and malignant characteristics of lung cancer. Aim II: To determine the relative contribution of ALDH-l and ALDH-3 to resistance against 4-hydroperoxycyclophosphamide (4-HC) in lung cancer cell lines. We will use the antisense RNA (AS) approach, and ribozymes as an alternative approach, in order to study the effects of downregulation of ALDH-1 and/or ALDH-3 on tumor cell growth and drug sensitivity. Significance Determining the expression pattern of these enzymes in primary lung cancer samples and contrasting it to the expression in normal and pre-malignant tissues will be critical to understanding the etiology for the overexpression of ALDH-l/ALDH-3 in NSCLC cells and designing a strategy to affect the natural history of these cancers. Being able to manipulate their expression by antisense RNA, ribozyme, or other factors will have significant impact on the treatment of these cancers.
  
  
  
  
  
• Satya Narayan, Ph.D. 
  The long-term goal of this clinical innovator award application is to determine the mechanisms by which secondhand smoking is associated with breast carcinogenesis. Increased gene mutation and compromised DNA repair are two of the many initiating events of smoke carcinogens-induced breast cancer development. However, the mechanism(s) by which DNA repair is blocked, mutations are accumulated, and normal breast epithelial cell transformation is initiated after exposure to smoke carcinogens is not clear. Mutations of the tumor suppressor adenomatous polyposis coli (APC) gene are known to occur in the early stages of colon cancer development, but the roles of altered expression of APC in extra colonic cancers such as breast cancer have not been clarified. It has been shown that a low-level expression rather than a mutation of APC is most common in breast cancers. The clinical significance of the low levels of APC in breast carcinogenesis is unknown, In our studies, we propose a highly novel concept where the decreased level of APC is responsible for the decreased DNA repair capacity, which is the underlying cellular mechanism(s) in smoke carcinogens-induced initiation and progression of transformation in normal breast epithelial cells. We hypothesize that in normal breast epithelial cells the wild-type APC maintains a binding-equilibrium with the DNA base excision repair proteins, proliferating cell nuclear antigen (PCNA) and apurinic/apyrimidinic endonuclease (APE). It helps in the process of smoke carcinogen-induced repair of abasic lesions and protects cells against genetic instability. In the absence of adequate levels of functional APC, the dynamic interaction of PCNA and APE at the abasic DNA is decreased, which, in turn, causes a decrease in DNA repair and an increase in mutations. These events lead to an increased genetic instability and the initiation and transformation of normal breast epithelial cells. We propose to establish the role of APC in DNA base excision repair and to determine its role in smoke carcinogens-induced breast carcinogenesis. Our in vitro model system will use the normal human breast epithelial cell lines in the presence or absence of treatment with cigarette smoke condensate and with benzo[a]pyrene, a well known cigarette smoke carcinogen. This is a highly innovative project, and for the first time, we will examine the molecular mechanisms underlying the functions of wild-type APC in DNA repair and breast carcinogenesis. This will guide us in an earlier diagnosis of smoking-induced breast carcinogenesis and help in the development of new chemotherapeutic agents for the treatment of this deadly disease.
  
  
  
  
  
• Venu Raman, Ph.D. 
  Breast cancer is the leading cause of death in women worldwide. It is estimated in the US alone, per year, 208,000 women will be diagnosed with breast cancer and 40,200 will die of this disease. Recently, several studies have reported a link between environmental tobacco smoke (ETS) and increased risk of breast cancer. As breast development is a dynamic process that responds to hormonal signaling and continues throughout the reproductive life of a woman, it is conceivable that aberrant signaling pathways can result in breast cancer progression. A challenge is to identify genetic alterations, following exposure to environmental tobacco smoke that predispose to breast cancer formation and progression. This knowledge will provide opportunities to design therapeutic agents to prevent cancer cells from disseminating. The specific aims of this project are 1) to identify gene expression patterns in normal mammary epithelial cell cultures (HMECs) following exposure to the key heterocyclic aromatic hydrocarbon chemical found in tobacco smoke, B [c]PDE-racemic anti-3 ,4-dihyroxy- 1,2-epoxy- 1,2,3 ,4-tetrahydrobenzo[c]phenanthrene, 2) to verify the effects of B[c]PDE target genes identified in mammary epithelial cells, and 3) as ETS has been shown to affect the spectrum of p53 mutations, we will study the effects of the chemotherapeutic agent tamoxifen on p53 expression. The Affymetrix mammalian cDNA chip array will be used to identify differential gene expression pattern in HMECs following exposure to B [c]PDE. Identified target genes will be transfected into immortalized normal mammary epithelial cells to establish their ability to induce neoplastic transformation. Finally, based on our preliminary results, we will ascertain if tamoxifen can regulate endogenous p53 expression in breast cancer cells. This is important, as the use of tamoxifen, an estrogen antagonist, in breast cancer patients with ER+/PR+ (estrogen receptor, progesterone receptor) is based on the observation that blocking ER functions can lead to decreased proliferation. However, recent evidence suggests that depending upon the cellular environment, use of tamoxifen can convert inactive ER and PR to their activated forms. This variant of the signaling pathway can be detrimental to the therapy protocol itself if the status of the p53 gene is not identified. The spectrum of p53 mutations in breast cancer patients exposed to tobacco smoke is enhanced, as compared to non-exposed subjects. Therefore, activating mutated p53 gene may result in an aggressive phenotype in breast cancer. Thus, deciphering the effects of environmental tobacco smoke exposure will provide us with clues to design better molecular therapeutic targets for breast cancer treatment.
  
  
  
  
  
• Barry Trink, Ph.D. 
  The most important cause of bladder cancer is cigarette smoke. Many of the toxins that enter the body after inhalation of cigarette smoke are absorbed into the bloodstream and excreted by the kidneys into the urine. Because urine stays in the bladder for hours before it is expelled, the bladder epithelium is exposed to prolonged contact with carcinogens absorbed from smoke. In the United States, tobacco use accounts for about half of all bladder cancers. The objective of this project is to clone the putative oncogene in the chromosomal region of 20q11 region and carry out critical functional studies to understand its role in bladder cancer. This region is implicated as a putative proto-oncogene locus in both familial and sporadic bladder cancer. Bladder cancer is the fourth most common internal cancer in American men, and its incidence is rising with each passing year. But early diagnosis can often cure the disease, and new therapeutic programs are improving the outlook for patients with advanced disease. In 1996, we described a case of a germline translocation t(5;2O)(pl5;q11) associated with familial transitional cell carcinoma (TCC) suggesting the presence of an oncogene or tumor suppressor gene in the affected region. In addition, we have evidence of 20q11 amplification in sporadic tumors. We will use the P1 clone covering the breakpoint area to clone the gene affected by this translocation. To date, we have discovered 3 candidate genes from this region. We will perform FISH analysis with this probe in primary bladder tumors and preneoplastic lesions to establish the frequency of amplification in bladder cancer progression. Finally, we will test for the specific activation of this putative oncogene by sequencing, Southern and Northern Blot analyses, and various functional studies. The identification of this novel oncogene could lead to new therapeutic strategies for bladder cancer
  
  
  
  
  
• Brian J. F. Wong, M.D., Ph.D. 
  Tobacco smoke produces a spectrum of changes in the delicate epithelial layers of the vocal cords, which may vary from benign processes such as chronic laryngitis to malignant and pre-malignant conditions such as dysplasia, carcinoma in situ, and frank invasive laryngeal cancer. Since it is extremely difficult to distinguish among these disorders a vocal cord (TVC) biopsy is needed. Biopsy requires microsurgery, which places the patient at significant risk for iatrogenic dysphonia. We propose to construct an Optical Coherence Tomography (OCT)-based device to image and differentiate early laryngeal cancers from benign laryngeal diseases without biopsy. OCT is a non-contact, emerging imaging modality that uses light to construct high-resolution (10 mm), cross-sectional images of tissue to depths of up to 3 mm. The objectives of this study are to: a) image the vocal cord microstructure in patients undergoing laryngeal microsurgery and obtain normative information on vocal cord cross-sectional microstructure; and b) compare OCT images with biopsies of the vocal cord in patients with early laryngeal cancer to determine how accurately OCT can diagnose early cancers. In preliminary investigations, we have used laboratory OCT devices to obtain high-resolution cross-sectional images of porcine vocal cords, and have identified key structures such as the basement membrane and in real time, have monitored chemical burns and simulated laser surgery. This study will enroll approximately 120 adult patients undergoing laryngeal microsurgery at UC Irvine Medical Center. Correlation of OCT images with conventional histology will allow determination of the efficacy of our device for diagnosing early vocal cord cancer. Morphometric measurements (e.g. thickness of epithelium, basement membrane etc.) on both optical and pathologic tumor sections will be made and compared as well. In addition to structural information on vocal cord microanatomy, the OCT device will provide functional images of specific tissue properties, such as birefringence (related to collagen content and degree of organization (Polarization-sensitive OCT) and microscopic blood flow images (using the Doppler technique or Optical Doppler Tomography). We expect our instrument to allow assessment of: 1) vocal cord basement membrane integrity; 2) basement membrane violation by epithelial tumors (which is the hallmark of invasive carcinoma); and 3) the true lateral and deep extension of vocal cord tumors, which will direct surgeons to biopsy regions with higher probabilities of cancer. Further, OCT imaging will provide a means of documenting changes in 3-D vocal cord microstructure over time.
  
  
  
  
  

Young Clinical Scientists

• Betsy J. Barnes, B.A., Ph.D. 
  The overall objectives of the current proposal are to investigate the relationship between expression of interferon regulatory factor 5 (1RF-5) and the tumor suppressor p53 in human non-small cell lung cancer, to determine the role of IRF-5 in these malignancies and to develop novel IRF-5-based therapies for lung cancer.
  
  Lung cancer due to cigarette smoking is a leading cause of death in the United States. Despite many years of research with conventional treatments, the majority of patients diagnosed with lung cancer eventually die from their disease. Mechanism-based therapies directed at tumor specific pathways offers hope for the development of novel treatments. Transcription factors of the interferon (TFN) regulatory factor family play essential roles in the regulation of genes induced by viral infection, immuno-stimulation as well as in cell growth regulation. IRF-5 is a newly identified transcription factor that was recently shown to be a direct target of p53 and may be involved itself in oncogenesis as a tumor suppressor. We have shown that expression of IRF-5 is absent in numerous leukemias, lymphomas and non-small cell lung carcinomas that may indicate a propensity for IRF-5 gene deletion or silencing. Studies in B cell lymphomas reveal that exogenous IRF-5 inhibits in vitro and in vivo tumor cell growth in the absence of wild-type p53. Results indicate that IRF-5 represses lymphoma cell expansion via induction of a G2/M arrest and apoptosis. Our studies have recently focused on non-small cell lung cancer since the majority of these malignancies express mutant, non-functional p53 or are lacking expression of IRF-5. The Specific aims of this proposal are: 1) To examine expression of IRF-5 and p53 in multiple human lung cancers. Human lung adenocarcinoma cell lines will be obtained from NCI, RNA will be isolated and transcripts analyzed by reverse transcription (RT)-PCR analysis and DNA sequencing. 2) To investigate the tumor suppressor potential of IRF-5 in the growth regulation of lung cancers where IRF-5 and/or p53 are not expressed. IRF-5 will be over-expressed in these tumor cells and cell-cycle progression analyzed. Cells will also be examined for apoptosis in the presence or absence of IRF-5. 3) To elucidate the precise mechanism through which IRF-5 induces tumor cell growth arrest, apoptosis or necrosis. We will examine IRF-5 induced genes using DNA microarray technology and the Ribonuclease Protection Assay (RPA). Ultimately, the final goal will be to establish a clinical regime for the use of IRF-5 as replacement therapy for patients with non-small cell lung cancer, specifically adenocarcinomas lacking wild-type p53 or IRF-5.
  
  
  
  
  
• Raka Bhattacharya, Ph.D. 
  Lung cancer is one of the most common causes of cancer deaths worldwide and the major risk factor for lung cancer is tobacco smoke exposure, which accounts for 70-80% of lung cancer related deaths. Understanding the molecular abnormalities that give rise to lung cancer associated with tobacco smoke exposure will provide valuable insight into the pathogenesis of this disease and may lead to new treatment strategies. We are studying the architectural transcription factors, HMG-I and -Y, which are encoded by the HMG-J/Y gene. Preliminary data from our laboratory suggests that the HMG-I/Y gene is an oncogene important in the pathogenesis of human lung cancer associated with tobacco smoke exposure. Specifically, HMG-I/Y proteins are overexpressed in several lung cancer cell lines, including those associated with tobacco smoke exposure. We also showed that HMG-I/Y proteins are critical to the transformed phenotype in lung cancer cells because decreasing these proteins using an antisense approach blocks transformation. Moreover, overexpression of the HMG-I or-Y isoforms leads to a transformed phenotype in experimental cell lines derived from lung cells. Thus, we hypothesize that HMG-I/Y is an oncogene that contributes to the development of lung cancer and the objective of this proposal is to explore the role of HMG-I/Y in lung cancer associated with cigarette smoke exposure.
  
  My specific aims are: 1.) Determine if HMG-I/Y and- C overexpression correlates with a more aggressive phenotype in human lung cancer using Northern, Western, RT-PCR, immunohistochemical, and microarray analysis. 2.) Determine if HMG-I/Y or -C proteins are essential for the transformed phenotype in the lung cancer cell lines using an antisense approach. 3.) Identify HMG-l gene targets involved in transformation in lung cancer using microarray analysis. 4.) Define the role of HMG-I/Y in chromosomal instability using spectral karyotyping (SKY) analysis.
  
  Results from this work will advance our knowledge of lung cancer associated with tobacco smoke exposure and should lead to new treatment strategies.
  
  
  
  
  
• Fred Bunz, M.D., Ph.D. 
  The objectives of this proposal are to determine the roles of the checkpoint kinases in the activation of p53, the protein product of the P53 tumor suppressor gene, and to devise a cell-based screen to isolate therapeutic compounds that inhibit these kinases. P53 inactivation occurs frequently in tumors, including those of the breast, the bladder, the lung, and the head and neck, and is considered to be an important etiologic factor in the development of many cancers.
  
  Over the past several years, the idea that loss of p53 function can alter cellular responses to therapeutic agents has become widely accepted. When challenged with a therapeutic agent that causes DNA damage, human cells respond by the activation of pathways that lead to cell cycle arrest and apoptosis. A central regulator of both of these pathways is the product of the P53 gene. In response to DNA damage, a signaling cascade of serine/threonine protein kinases causes the stabilization and activation of the p53 protein, which in turn induces the transcription of downstream genes that effect cell cycle checkpoints and trigger apoptosis. The checkpoint kinases Chkl and Chk2 are known to phosphorylate p53 following DNA damage, but the specific roles of these kinases and the consequences of these modifications upon p53 function remain incompletely understood. The specific aims of this project are: 1) To develop a model system for the genetic analysis of Chkl and Chk2 function in human cells. This model system will be created by the homozygous disruption of the CHK1 and CHK2 genes in human cell lines that have wild type p53 function. 2) To determine the effects of CHK1 and CHK2 disruption on p53 stabilization and the cellular response to DNA damage. Using gene-targeted cells, the precise mechanistic roles of Chkl and Chk2 in the implementation of cell cycle checkpoints will be examined in detail. It will be determined how the checkpoint kinases may play a role in p53-dependent apoptosis. In addition, the roles of CHK1 and CHK2 in the maintenance of chromosomal stability will be assessed. 3) To devise a cell-based screen to identify small molecules that can specifically target Chkl and Chk2 kinase activity. In the long term, the gene targeted cell lines described here will be evaluated as tools to evaluate novel kinase inhibitors with therapeutic potential.
  
  
  
  
  
• Robert L. Ferris, M.D., Ph.D. 
  The main etiologic factor contributing to head and neck squamous cell carcinoma (HNC) is tobacco-carcinogen exposure. For several reasons, the immune system is not able to mount an effective anti-tumor response, despite a large number of tumor-specific genetic alterations that should enable distinction by the immune system between normal and cancerous tissues. The ability to subvert immune cell function by human tumors, including HNC, leads to host immunosuppression and may facilitate tumor progression. Recent evidence indicates that HNC can interfere with the survival of T cells, as measured by increased rates of cell death and rapid turnover of circulating lymphocytes. In addition, functional impairment of peripheral and infiltrating lymphocytes has been increasingly demonstrated in such patients. Peripheral T cells of patients with HNC, but not of normal individuals, appear to die by apoptosis, mediated in part by members of the TNF family of receptors and ligands. Such rapid turnover of immune cells likely contributes to the failure of antitumor functions of the immune system. Moreover, immune-based therapies, including antitumor vaccines, are unlikely to be effective in patients whose lymphocytes undergo rapid apoptosis or are functionally impaired. We have also observed that a number of specific immune parameters, that are abnormal in tumor-bearing patients, can return to normal in patients after curative oncologic therapy. Thus the ability to prevent or arrest this rapid lymphocyte turnover has obvious therapeutic implications. We hypothesize that tumor antigen-specific T cells are preferentially targeted for early apoptosis, leading to host tolerance to the tumor. Based on extensive preliminary data, we propose to investigate the dynamic basis for T cell depletion in patients with HNC. In a prospective series of patients, this analysis will include the mechanistic study of this phenomenon, its in vivo kinetics, and therapeutic strategies for preventing early death of circulating immune cells. Identification of subsets of effector T cells preferentially destined to die in these patients' circulation will be performed using tetrameric antigen-loaded MHC molecules and selected biomarkers of apoptosis. In addition we will directly study the fate of circulating T lymphocytes in HNC patients and in age-matched healthy donors, using in vivo cell labeling with nonradioactive deuterated glucose as well as with ex vivo T cell receptor excision circle (TREC) measurements for naïve T cell replenishment. Our research into the reversal of this tumor-specific lymphocyte death may be critical to the successful application of immune based therapy for cancer.
  
  
  
  
  
• Robert F. Foronjy, M.D. 
  Plasma phospholipid transfer protein (PLTP) is a protein whose best studied function is to shuttle phospholipids amongst lipoprotein particles in the circulation. It is also known to be able to affect the concentration of these lipoproteins and, in so doing, can impact on the development of atherosclerosis in hyperlipidemic mice. Recent data has shown that the principal site of activity of this enzyme is in the type II pneumocyte within the lung. This activity has been shown to be increased in tissue samples and induced sputum samples from emphysema patients compared to controls. Given the effects of this protein on phospholipid metabolism, it appears probable that this protein may have significant effects on surfactant composition. Mice deficient in PLTP activity have been shown to have alterations in the concentration of surfactant-associated proteins in their lungs. There is significant evidence to suggest that disturbances in surfactant function may impact negatively on the course of chronic obstructive pulmonary disease (COPD). Surfactant associated proteins A and D are known to have several important functions within the lung. In addition to interacting with surfactant and stabilizing airways, they have important antimicrobial and anti-inflammatory functions that may be important in the development of smoking related diseases. We hypothesize that PLTP may impact significantly on the development of smoke related lung disease by modulating the composition and concentration of surfactant proteins and phospholipids within the lung. The goal of this project is to determine if PLTP activity measured from the induced sputum of COPD patients correlates with disease severity and progression in these patients and whether it may serve to identify those individuals more susceptible to the toxic effects of passive smoke exposure. This will be assessed by performing serial induced sputum analysis on a group of COPD patients and correlating decline in lung function over time with levels of PLTP activity. To determine if passive smoke exposure alone is sufficient to induce PLTP activity, levels obtained from COPD patients will be compared to healthy non-smoking controls and individuals with significant second hand smoke exposure. If PLTP activity were found to correlate significantly with disease progression and to be induced by passive smoke exposure, it would have tremendous diagnostic and therapeutic implications for this disease. For one, it would allow clinicians to know with better certainty which patients are more predisposed to developing lung disease from cigarette smoke exposure. This would allow physicians and others to know which patients need more aggressive monitoring and care. In addition, the insight provided from this study may provide clues into the nature of this disease that would open up new avenues of potential treatments.
  
  
  
  
  
• Maureen R. Horton, M.D. 
  Chronic bronchitis, due to passive or active smoke exposure, afflicts more than 14 million Americans and consumes 11 billion healthcare dollars a year. To date, the exact cellular and molecular mechanisms accounting for chronic bronchitis have yet to be elucidated. At the cellular level, the hallmarks of chronic bronchitis are mucus gland hypertrophy with excess mucus production, accumulation of inflammatory mediators, inflammation of the airway walls, and breakdown and turnover of the extracellular matrix. We hypothesize that the extracellular matrix is not only a target of destruction in chronic bronchitis, but that it plays a critical role in perpetuating the unremitting inflammation characteristic of this disease. Specifically, we propose that the extracellular matrix component Hyaluronan (HA) mediates the inflammation of chronic bronchitis through the induction of cytokines, chemokines and modulatory enzymes by epithelial cells.
  
  The overall objective of this proposal is to define the cellular and molecular mechanisms mediating HA-induced inflammatory gene expression by airway epithelial cells with the ultimate goal of identifying pharmacologic inhibitors and potential treatments. We will pursue this goal with the following specific aims: (1) Define the cellular mechanism responsible for extracellular matrix-induced IL-8 and IP-10 in airway epithelial cells. We will evaluate the roles of new protein synthesis, autocrine feed back loops, rnRNA stability and transcriptional regulation by HA. Additionally, we will further identify the profile of chemokines, cytokines and modulatory enzymes induced by extracellular matrix in epithelial cells. (2) Define signal transduction pathways and cis/trans-acting elements responsible for extracellular matrix-induced inflammatory genes in epithelial cells. We will accomplish this aim by using inhibitors of signal transduction, electrophoretic mobility shift assays and transient transfection assays with promoter driven reporter constructs for IL-8 and IP-10. Additionally, we will investigate the role of coactivators in HA-induced chemokine expression in epithelial cells. (3) Identify specific pharmacologic inhibitors of extracellular matrix-induced epithelial cell expression of inflammatory genes and the ability of CXC chemokine receptor inhibitors to abrogate HA-induced IL-8 and IP-10 expression in epithelial cells and subsequent neutrophil chemotaxis.
  
  
  
  
  
• Chien-Fu Hung, Ph.D. 
  Human papillomavirus (HPV) infections, particularly type 16 and 18, have been associated with cervical intraepithelial neoplasis (CIN) and cervical cancer. In screened women, persistence of HPV infection has been found to be associated with a greatly increased risk of developing CIN. It has been also been suggested that smoking may be the most important factor affecting the progress of CIN after HPV infection.
  
  Current primary therapies for cervical cancer include radical surgery and chemoradiation. However these modalities are associated with significant treatment toxicity, and overall survival remains a dismal 40%. Antigen-specific immunotherapy provides an ideal alternative approach for the control of cancer over the current therapies such as chemotherapy and radiation therapy because it can eradicate systemic tumor harbored at multiple sites in the body, as well as the specificity to discriminate between malignant and normal cells.
  
  In treating cervical cancer, several human papillomavirus (HPV) E7-specific DNA vaccines have been shown to treat established E7-expressing murine tumors in animals. Moreover, the use of DNA vaccines for generating antigen-specific CD8+ T cell responses has been well established in animal models. However, little is known about the quantitative and qualitative aspects of CD8+ T cell responses and protective immunity generated after repeated DNA vaccinations.
  
  In this FAMRI proposal, we hypothesize that there may be quantitative and qualitative difference of CD8+ T cells generated in human receiving increasing number of DNA vaccinations. We also hypothesize that human patients receiving an increased number of pNGAVL4a-Sig/E7(detox)/HSP70 DNA vaccinations will exhibit higher E7.-specific cytotoxic T lymphocyte (CTL) activity and a rapid expansion of E7-specific IFN-g-secreting CD8+ T cells which may lead to a stronger therapeutic effect against HPV- 16+ CIN3 lesions.
  
  Using the blood sample taken from our patients receiving the regimen of pNGAVL4a-Sig/E7(detox)/HSP70 DNA vaccines at 3 different stages (i.e. before vaccination, after receiving 2 shots, and after receiving all 4 shots of pNGAVL4a-Sig/E7(detox)/HSP70 DNA vaccine), we want to characterize the quantitative and qualitative differences in the CD8+ T cells using four immunological assays, an avidity assay, and a microarray analysis. We will attempt to correlate the data gathered in the immunologic analysis to the clinical therapeutic outcome. Furthermore, we will compare the gene expression profiles of the CD8+ T cells in an attempt to identify the key expression factors contributing to the qualitative difference of CD8+ T cells generated by the vaccine at different stages of the regimen.
  
  
  
  
  
• Edgar A. Jaimes, M.D. 
  Cigarette smoking has been identified as the most important source of preventable morbidity and mortality in the U.S. Cigarette smoking constitutes a major risk factor for atherosclerotic cardiovascular disease and pulmonary hypertension. In spite of the widely appreciated magnitude of the problem, there are still critical gaps in our knowledge of the mechanisms involved. Endothelial dysfunction is one of the first events in the pathogenesis of atherosclerosis and pulmonary hypertension and is manifested as impaired endothelium dependent relaxation (EDR) to Nitric Oxide (NO). Clinical studies have demonstrated that cigarette smoking is associated with impaired NO mediated EDR. The mechanisms involved are multiple and not completely understood although accumulating evidence suggests that superoxide anion (O2-) may play a critical role. Our main long-term goal is to determine the mechanisms by which cigarette smoke promotes cardiovascular disease in smokers. To attain this goal we will pursue the following specific aims: 1. To identify the mechanisms by which cigarette smoke produces endothelial dysfunction. First we will determine whether cigarette smoke in vitro upregulates the expression and activity of enzymes known to produce O2 such as NADH/NADPH oxidase and xanthine oxidase in cultured human vascular cells. We will use real time-PCR, western blot and O2 measurements to determine the expression and activity of these enzymes. Second, we will determine the role of these enzymes activity on cigarette smoke induced endothelial dysfunction by determining the effect of O2 produced by these enzymes on NO bioactivity and NO synthase activity. 2. Our second aim is to determine whether as a result of smoking the vessels from smokers have a phenotypic change that results in increased baseline production of 02 and the mechanisms involved. We will measure the ex vivo production of O2- in vessels from the systemic and the pulmonary circulation of smokers and non-smokers as well as in vascular cells cultured from these vessels. Using real time -PCR and western blot we will determine the expression of NADH/NADPH oxidase and xanthine oxidase in the same vessels and cells. These studies will increase our understanding of the mechanisms for cigarette smoke induced endothelial dysfunction and increased cardiovascular risk in smokers.
  
  
  
  
  
• Saeed R. Khan, M.S., Ph.D. 
  Breast cancer is the most common form of cancer in women and second leading cause of cancer deaths in women in the United States. Although major advances have been made in early detection, prevention, and treatment of early disease, the need for more effective therapy in the fight of late stage breast cancer continues. The mouse double minute 2 (MDM2) oncogene has been suggested as a target for breast cancer therapy. It is amplified: or overexpressed in human breast cancer and MDM2 levels are associated with poor prognosis of human breast cancer. It has been shown that MDM2 has a role in tumor growth through both p53-dependent. and p53-independent mechanisms. We will be investigating the potential value of MDM2 as a drug target for breast cancer therapy by inhibiting MDM2 expression with specific boronic-chalcone analogs. Such an approach should provide a basis for development of anti-MDM2 boronic-chalcones as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics. A class if anticancer agents such as chalcones, has shown promising therapeutic efficacy for the management of human cancers. Recent studies have shown that anticancer agents such as chalcones induce apoptosis in variety of cell types, including breast cancers. However, these carboxylic acid analogs of ehalcone are equally toxic to normal cells. In this proposal, a boronic-chalcone strategy in outlined that will overcome this limitation. Boronic acid chalcone analogs should be more cytotoxic to the breast cancer cells and not to human normal cells. since boronic acid is an isostere of carboxylic acid, boron substituted for a carboxyl carbon at the reactive substrate site can bind strongly but reversibly with electron donating groups of the MDM2 proteins. Based on this mechanism, boronic acid chalcone analogs should be more cytotoxic to the breast tumor cells than carboxyic acid analogs of chalcones. These boronic-chalcone analogs, therefore, represent a potentially novel approach toward the treatment of metastatic breast cancer. The goal of the current proposal is to build upon the paradigm of the boronic-chaleone analogues to determine if more effective and breasttumor selective agents can he found. Upon the identification of active compounds, these cvtotoxic agents will be tested for their potency arid selectivity for breast tumor cells. The lead compounds will then be tested in vivo models of human breast cancer. These studies will serve to identify the best candidate that will subsequently be tested in clinical trials as treatment for metastatic breast cancer.
  
  
  
  
  
• Andreea A. Nanci, M.D. 
  Background: Cigarette smoking increases the risk of pancreatic cancer. The treatment of this very aggressive disease has advanced little with essentially a 100% mortality rate. Thus, the need for novel treatments is obvious. Cancer immunotherapy represents such a novel approach. Dendritic cells (DC), as the most potent antigen presenting cells (APC), play a central role in the initiation of antigen- specific immune responses. These cells can efficiently phagocytose both apoptotic and necrotic cellular debris. However, controversy exists with respect to which type of material is optimal for the induction of the "mature" DC phenotype and crosspriming. Apoptotic material appears to be more efficient at cross-priming and necrotic cellular debris more potent at induction of maturation. Given these contrasting effects, it is reasonable to expect differences in the signaling pathways of DCs when they encounter these two different types of cellular debris. It is important to understand the effects of DC exposure to each type of debris, apoptotic or necrotic, in order to develop more efficient immunotherapeutic strategies and enhance DC function with respect to processing and presentation of antigens.
  
  Central Hypothesis: Human monocyte-derived DCs utilize different signaling pathways in response to apoptotic or necrotic cellular debris.
  
  Objective/Specific Aims:
  
  I will test the central hypothesis by pursuing the following three Specific Aims:
  
  1. Evaluate the role of. putative receptors, eg. CD91, TLR 2&4, CD36, and PSR, along with established downstream signal cascade molecules in the uptake of matched apoptotic or necrotic tumor material.
  
  2. Evaluate alternative signaling pathways involved in responses to the uptake of matched apoptotic or necrotic tumor material.
  
  3. Characterize the activation and 'maturation' state of DCs, including tumor and antigen-specific T
  cell stimulatory capacity after uptake of matched apoptotic or necrotic tumor material
  
  Methods: Photodynamic therapy (PDT) has been shown to have the capacity to induce either apoptotic or necrotic cell death in vitro and in vivo. Thus, this technique represents a useful tool and will be used to induce differential cell death in 3 dimensional cell structures (spheroids) derived from a HLA-A2 expressing human pancreatic cancer cell line. Human monocyte-derived DCs will be exposed to these preparations. Antibodies targeting CD91, CD36, the phosphotidylserine (PSR) and toll like receptors (TLR) will be used initially to discriminate differential activation of these pathways. Additionally, we will use signal pathway selective DNA gene arrays to elucidate differences in these and alternative signaling pathways activated by exposure to matched apoptotic or necrotic cellular debris. DC cell maturation and activation phenotype will be assessed by Flow Cytometry, Ribonuclease Protection Assay and in-vitro T cell immune responses including MEC tetramer analysis.
  
  Relevance: These studies will not only characterize the signals transduced in DCs when apoptotic or necrotic cellular material is encountered but will also lead to the establishment of optimal parameters for PDT induced cell death. Ultimately, this data will provide a basis for appropriate manipulation of DCs and help in the rational design of clinical trials combining PDT with DC- based immunotherapy for pancreatic cancer.
  
  
  
  
  
• Ben H. Park, M.D., Ph.D. 
  Breast cancer is a heterogeneous genetic disease but a common feature of all breast cancers is that they occur predominantly in women. Though the reasons for this are not entirely known, clearly a correlative relationship between estrogen exposure and an increased risk of breast cancer has been well documented. The focus of this study is to elucidate the mediators of estrogen receptor signaling as it relates to breast carcinogenesis. Understanding these pathways is of critical importance, as it would enable us to identify potential mechanisms of drug resistance, as well as aid in the identification of new targets for drug therapy. Although much insight has been gained into the biologic function and regulation of estrogen receptors, pathogenic mechanisms of how estrogen drives tumorigenesis remain elusive. For example, it is known that breast epithelial cancer cell lines expressing estrogen receptor alpha (ER alpha), proliferate in response to estrogen treatment, while estrogen antagonists induce a growth inhibitory response. However, cDNA overexpression of ER alpha in estrogen receptor negative breast epithelial lines induces growth inhibition upon treatment with estrogen, and this effect is reversible with pure estrogen antagonists. This negative growth effect of estrogen is also seen when these cell lines re-express their endogenous ER alpha after treatment with 5-aza-2'deoxycytidine and histone deacetylase (HDAC) inhibitors. Using cell-cell fusion techniques, we will fuse a normal breast epithelial line engineered to overexpress ER alpha, with the hormonally responsive ER alpha positive breast cancer cell line MCF-7. Resulting hybrid cells will be tested for their ability to respond to estrogens and anti-estrogens to determine which phenotype, cell proliferation versus growth inhibition, is dominant. With this information, eDNA libraries will be constructed from the parental line that displays the dominant phenotype. Functional cloning will then be employed using the recessive parental cell line as the recipient, to isolate the factors that mediate the MCF-7 cell line's ability to respond positively or negatively to estrogen or anti-estrogens respectively. These genes will then be examined by targeted gene disruption and/or overexpression in our normal ER alpha overexpressing breast cell line to confirm that they can switch the cell's response to estrogen from growth inhibition to cellular proliferation. These genes would then define mediators of ER alpha's ability to stimulate growth in breast cancer cells and therefore represent new targets for drug therapy.
  
  
  
  
  
• Edward S. Peters, DMD, SM, ScD 
  The Young Clinical Scientist Award from the Flight Attendants Medical Research Institute would provide the initial support to establish Dr. Edward Peters as a new and independent young investigator in cancer epidemiology with a specific focus on understanding the association between tobacco exposures and cancers of the upper aerodigestive tract. Epidemiologic evidence has clearly demonstrated that the principal etiology of oral cancers are tobacco and alcohol consumption, with over 75% of all oral cancer in the United States attributable to these two avoidable risk factors. At the same time, only a relatively small proportion of exposed individuals develop this disease. Recent reports have revealed an alarming increase in the incidence of oral cancer among younger individuals, many without the established risk factors of alcohol or tobacco. Several environmental risk factors have been hypothesized to be responsible for the development of HNC in these individuals. However, none yet have been proven the cause of the disease.
  
  Individual genetic susceptibility may have a role in explaining variation in the patterns of occurrence of exposure-related cancers. Much of the research in this area has focused upon the modification of an exposure-response relationship by polymorphic genes that code for enzymes that either activate or inactivate xenobiotic compounds known to be associated with disease occurrence. Polymorphisms in many such genes have been identified and associated with an increased risk for lung cancer, particularly increasing susceptibility at low levels of tobacco exposure. Since tobacco carcinogens are clearly associated with oral cancer, further investigation into the role that genetic susceptibility has in oral cancer development is warranted, with particular attention to low exposure levels.
  
  We believe that we are uniquely situated to study the environmental tobacco smoke (ETS) - oral cancer relationship. Using oral cancer patients and population-based controls enrolled in a funded and ongoing case-control study of oral cancer (ROl CA78609) as study subjects, we propose to investigate the association between ETS and oral cancer. In particular we plan to examine how this relationship may be modified by genetic polymorphisms of carcinogen detoxifying enzymes that have been demonstrated to affect ETS-associated lung cancer risk.
  
  Specific aims to be addressed in the proposed study are:
  
  1. To develop an ETS exposure matrix to estimate the degree of ETS exposure in study participants
  
  2. To test the hypothesis that the risk for oral cancer among "younger patients" may be explained by the effects of ETS.
  3. To test the hypothesis that an association between ETS exposure and oral cancer is elevated among individuals with deletion in the glutathione S-transferase (GST) theta gene (GSTTI), or with deletion in the GST class mu (GSTMI) gene.
  
  4. To test the hypothesis that additional genetic susceptibility markers that will be considered in the parent study (e.g. CYP1A1, NAT2) modify the association of oral cancer and ETS exposure.
  
  5. To provide the framework in which ETS exposure can be included in subsequent hypothesis testing. For example, we plan to investigate the association between tobacco use and somatic alterations of oral cancers; funding this proposal will allow us to more carefully evaluate the role of ETS in shaping this relationship.
  
  
  
  
  
• Debabrata Saha, Ph.D. 
  This YCSA proposal describes a 2 year training program for Dr. Saha’s transition to scientific independence in the field of translational research. The candidate is a senior research fellow in the Department of Radiation Oncology. He will now expand on his scientific skills and knowledge base through multidisciplinary interaction with Dr. Hak Choy, Vice Chairman of the Department of Radiation Oncology which provides an ideal environment for this training program. It incorporates expertise from a broad array of disciplines and has extensive resources that can be used to support young faculty members, Dr. Saha is going to devote xx% of his time to pursuing his academic research goals during the award period.
  
  This research plan focuses on the role of combined therapy of radiation and specific targeted drugs in the human lung carcinoma cell lines. Throughout this study, he will focus on the regulation of Cyclooxygenase-.2 (COX-2) in response to the combined treatment of Flk-l antagonist (antiangiogenic drug), SU11248, and radiation. He will also extend his studies to evaluate the efficacy of a triple combination of specific COX-2 inhibitor celecoxib, SU11248 and radiation in this human lung carcinoma model. The background and preliminary data suggest that both Flk-l antagonists and the selective COX-2 inhibitor significantly enhance radiosensitivity in the human lung carcinoma model. The specific aims include: 1) To study the combined treatment of Flk-l antagonists and radiation in the regulation of COX-2 expression in the in vitro human lung carcinoma model. 2) To develop a rational for triple therapy strategies consisting of radiation and systematically administered angiogenic antagonists and COX-2 inhibitor in in vitro models for clinical testing. In order to perform these specific aims, Dr. Saha has outlined a research career development plan that includes a focused training period in the laboratory of Dr. Choy along with course work in relevant areas of radiation biology. Dr. Saha's long term goal is to make significant contributions to the understanding of combined modality therapy. It is expected that during this award period, Dr. Saha will progress toward his goal of becoming an independent scientific investigator in the field of translational research.
  
  
  
  
  
• Hamisu M. Salihu, Ph.D. 
  Background and Objectives Recent reports suggest that prenatal second-hand smoking may have negative consequences on the developing fetus although the results remain inconclusive. The main reason for the inconsistency is that the majority of these investigations used self-report rather than a biologic marker to establish exposure status. To date, only six published studies worldwide (5 in Europe and 1 in the US) have used a biomarker to establish the effects of prenatal second-hand smoking on fetal body growth. Also, there has been no study yet assessing the impact of prenatal exposure to second-hand smoking on brain size, a very important endpoint since it has been established that a small brain size at birth is highly predictive of subsequent neurobehavioral abnormalities, and also recent studies are suggestive that active maternal smoking may affect the growth of the fetal brain. In this proposal, we intend to study the effect of prenatal exposure to second-hand smoking on fetal body and brain size using maternal serum cotinine concentrations to establish exposure status among actively non-smoking gravidas. A second objective is to investigate the interaction between tobacco smoke exposure and maternal folic acid status in the pathogenesis of fetal body and brain growth reduction. This is important because there is data suggesting that oxidizing agents in tobacco smoke reduce the levels/activity of folic acid among smokers. To our knowledge, this has not yet been studied among pregnant women exposed to tobacco smoke. Since folic acid is known to promote optimal fetal growth in utero, it is important to determine the relationship.
  
  Research Design and Methods We will utilize existing data and blood samples obtained prenatally and at delivery from a cohort of pregnant women for this study. We will identify passive smokers from among actively non-smokers by measuring cotinine levels in maternal sera. We will also measure folic acid blood levels. We will estimate the effect of passive smoking during pregnancy on fetal body and brain size using logistic regression models for categorical outcome and multiple regression models for continuous outcome measures. The relationship between maternal folic acid levels and cotinine levels will be explored using Spearman rank correlation. This study will provide baseline data for future follow-up in adulthood to determine the relationship between passive smoking and cognitive and neuro-behavioral outcomes.
  
  
  
  
  
• Robert E. Walter, M.D., MPH 
  Chronic Obstructive Pulmonary Disease (COPD) affects more than 14 million of Americans, extorting a significant toll in both personal and financial terms. Despite tobacco control efforts, COPD is likely to remain a significant public health burden. The current paradigm for the pathogenesis of COPD involves the upregulation of inflammation and oxidative stress, usually in response to tobacco exposure, which alters the protease/antiprotease balance. The foundation for this model was laid forty years ago; in the intervening years, only limited progress has been made in improving our understanding of the mechanisms that lead to chronic airflow obstruction. Improved understanding of these molecular mechanisms that are associated with the decline in lung function will allow for targeting of therapeutic interventions, development of surrogate outcomes for clinical trials, and focusing public health interventions on those most at- risk for disease development.
  
  Several biomarkers of disease activity, including markers of inflammation, of matrix turnover, and of oxidative stress, have been inconsistently associated with COPD. We hope to improve our understanding of the relation of biomarkers of inflammation and oxidative stress to loss of lung function using two distinct approaches- (1) examining the relation of reported biomarkers in urine and serum to cross-sectional lung function and longitudinal decline of lung function among participants of the Framingham Heart Study, and (2) examining the relation of reported AND novel biomarkers from urine, serum, exhaled breath, and exhaled breath condensate to the diagnosis of COPD in the context of a case-control study. The case-control format will allow us to develop novel biomarkers, which we could then later validate by returning to the Framingham Heart Study cohort to examine the relation of these novel biomarkers to both cross-sectional and longitudinal rate of decline of lung function.
  
  
  
  
  
• Jonathan P. Winickoff, M.D., MPH 
  Overview. Jonathan P. Winickoff MD, MPH, an academic pediatrician, is applying for a Young Clinical Scientist Award to develop and enhance the skills necessary to become a successful independent investigator in the field of tobacco control. His long-goal is to develop a multidisciplinary research program dedicated to reducing child exposure to secondhand tobacco smoke and its associated diseases.
  
  Research Plan The goal of this research plan is to develop and test an office system to enhance the delivery of evidence-based secondhand smoke reduction strategies and tobacco treatment services for parents of children seen in pediatric practice. In three phases, the research plan uses exploratory, descriptive, and intervention designs to address parental tobacco control in the pediatric outpatient setting. Each phase of the research builds on what has been learned in the prior phase. Phase I is an exploratory study consisting of the preliminary adaptation of an evidence-based tobacco control strategy to the pediatric outpatient setting. Phase 1 is grounded on the Solberg and Wagner theoretical models, multiple interviews with each of several leaders in the field of outpatient preventive services implementation, and iterative adaptation of the proposed tobacco control office system based on these key interviews. Phase 2 is a qualitative study using focus groups from 8 pediatric practices to elicit pediatrician and key staff responses, barriers, and solutions to implementing the proposed tobacco control office system. Phase 3, an intervention study, examines the feasibility and efficacy of implementing within the pediatric office setting the tobacco control system that was developed in Phase I and refined in Phase 2. The design is a before-after design with one practice serving as the control. Outcomes include the institution of rules prohibiting smoking in homes with children and the enrollment of identified parental smokers into centralized tobacco control programs increasingly available throughout the United States. Results should justify a larger trial with randomization at the practice level. In summary, the research plan will provide critical information about the delivery of evidenced-based secondhand smoke reduction strategies within pediatric practice, and crucial experience for Dr. Winickoff in qualitative data analysis, survey methods, primary data collection, database management, statistical analysis, and intervention research.
  
  
  
  
  

Other Grants

• Martin C. Mahoney 
  A. Specific Aims
  
  1. Overview: Exposures to secondhand tobacco have been linked to lung cancers, heart disease and a variety of upper respiratory tract conditions. In recent years there has been increased emphasis on implementing tobacco control policies to reduce exposures to tobacco smoke exposure in work site, restaurants and other public settings. Despite notable legislative successes, little is known about whether these policy changes have resulted in reduced secondhand smoke exposures.
  
  While smoking represents an important risk factor for developing chronic obstructive pulmonary disease, this condition also occurs among former and never smokers. There is limited information regarding subtle changes in airways resistance resulting from exposures to secondhand smoke. Moreover, specialized techniques for detecting cytogenetic abnormalities could prove useful in documenting the genotoxic effects of secondhand smoke.
  
  This proposal outlines a plan to conduct a study examining current sources and levels of secondhand smoke exposures among non-smokers using validated measurement tools and assess blood cotinine measurement. The proposed methodology will combine interview data on tobacco smoke exposures with objective measures of urinary cotinine levels to explore relationships between secondhand smoke exposure and 1) measures of preclinical obstructive changes in lung function (e.g., airways resistance), 2) symptoms of upper respiratory tract/eye irritation, and 3) cytogenetic changes. Participants will complete a baseline set of measures (interview data, pulmonary testing, biologic specimens), then return for repeat assessment twelve months following the initial visit to examine shifts from baseline over this interval. It is anticipated that these results will yield significant contributions toward developing a better understanding of airway obstruction and its correlates among nonsmokers, as well as further characterizing the untoward health effects results from secondhand smoke.
  
  2. Objectives: The overall goal of this project is to assess secondhand smoke exposures among non-smokers and examine associations between secondhand smoke and preclincial changes in lung resistance and premalignant cytogenetic abnormalities as assessed using peripheral blood specimens.
  
  This study will examine several hypotheses:
  
  1) Non-smokers continue to be exposed to secondhand tobacco smoke based on interviews and continine levels;
  
  2) Higher levels of secondhand smoke exposure/cotinine levels will be correlated with increased reports of upper airway/eye irritation;
  
  3) Higher levels of secondhand smoke exposure/cotinine levels will be correlated with deceased parameters on lung function testing
  
  4) Higher levels of secondhand smoke exposure/cotinine levels will be correlated with deceased parameters on lung function testing;
  
  5) Among non-smokers with normal spirometry testing, higher levels of secondhand smoke exposure/cotinine levels will be correlated with increased airways resistance detected via body plethysmography; and,
  
  6) Subject with higher levels of secondhand smoke exposure/cotinine levels will exhibit increased rates of cytogenetic abnormalities.
  
  Data collected from non-smoking participants will include a baseline face-to-face interview to assess exposures to secondhand tobacco smoke, tobacco use history, focused medical history, including information on reactive airways disease, atopy/environmental allergens and upper respiratory symptoms, as well as demographic information. Subjects will complete pulmonary function testing (spirometry with possible plethysmography) and consent to the collection of urine to assess cotinine levels and blood to examine for the presence of cytogenetic changes. Interval assessments, which will occur 12 months after initial data collection, will repeat all measures and examine interval changes in secondhand smoke exposures and corresponding changes in lung function parameters and cytogenetic markers of cellular injury/risk.
  
  
  
  
  
• Justin D. Pearlman, M.D. 
  We propose to establish a multi-disciplinary team of Cardiologists, Oncologists, Pulmonologists, Physiologists, and Engineers to focus on parallel efforts for the accelerated development of novel methods for early accurate detection of major afflictions of personnel who have been subjected to secondhand smoke (aka environmental tobacco smoke, or ETS). Those inflictions include inflammation, ischemic heart disease, and cancers. We have identified specific novel opportunities to make significant advancements in early accurate diagnosis and treatment guidance for the major causes of death and disability in this group deserving detailed investigations that would be enabled by this requested funding.
  
  Medicine has long focused on catastrophe intervention - dramatic responses to major afflictions such as heart attack or advanced cancer. Our goal here is to identify much earlier stages of disease and determine how to prevent or reverse the damage and avoid the consequent catastrophes. Thus we plan to develop early detection and guidance for minimally invasive intervention. Early detection, before the damage is permanent, has the advantage touted in the adage "a stitch in time saves nine”. We know that those who have been exposed to secondhand smoke have incipient illnesses long before they become aware of them, and that the leading causes of death in that group are cardiovascular disease and cancer. Just as these two disease categories have a common cause, secondhand smoke, they also have a very important pathophysiology in common: small vessel remodeling (microvascular changes). Although these vessels are too small to see by conventional x-ray, we have invented potential means, to identify and characterize these microvascular changes using new technology. We have preliminary data that demonstrate tremendous success of this approach in guiding a new treatment of heart disease called therapeutic angiogenesis (stimulation of new vessel development to feed hungry regions). We now propose to expand that success into new disciplines, to use success in one area to achieve new goals, to provide novel approaches for early diagnosis and to guide cures of diseases related to secondhand smoke / ETS.
  
  We have shown that the strategy of imaging microvascular changes, with preliminary application to early cancer identification, (N=1), can identify a 1 non-bronchogenic carcinoma that would otherwise have been missed for at least a year; whereby the early detection led to a cure, entirely avoiding a need for chemotherapy. That is not the level of preliminary data that would suffice for NIH funding, nor are all the engineering and clinical methods optimized - but that is exactly what makes this an excellent opportunity for FAMRI to support the discovery and development of novel methods for early accurate diagnosis and cures. There is a firm biological basis for this with strong potential to dramatically impact on early diagnosis and cure of major diseases afflicting those exposed to secondhand smoke.
  
  Work can proceed on the usual slow track (small incremental improvements reimbursing known methods in a sequence of grants), but that likely would not have clinical results or major advances in time to help the current generation of class members who are at risk. We therefore propose a fast track of parallel efforts expanding on microvascular imaging and molecular -imaging applications to the areas of concern for class members: for early detection of a range of specific cancers, and to characterize the risk to the heart relating to secondhand smoke (ETS). The methods can subsequently be exported to central hubs for wide access to the technology advance.
  
  
  
  
  
• Richard A. Robbins, M.D. 
  Specific Aims:
  
  I . To assess lower respiratory tract inflammation in subjects directly exposed to environmental tobacco smoke
  
  2. To assess lower respiratory tract inflammation in subjects exposed to passive environmental tobacco smoke.
  
  3. To non-invasively assess individuals at risk to develop emphysema from environmental tobacco smoke.
  
  Background: Neutrophil accumulation in the lung is a prominent feature of smoking-induced lung disease secondary to environmental tobacco smoke (ETS). Current concepts suggest that activation of neutrophils to release proteases and oxygen-derived free radicals contribute to the pathogenesis of the disease. An important step is the local generation of neutrophil chemoattractants within the lung resulting in the attraction of neutrophils.
  
  Exposure to ETS can result in COPD. However, only about 10-20% of smokers develop COPD and nonsmokers passively exposed to ETS develop COPD at about double the rate of the ETS unexposed population. Genetic factors have been postulated to play a role, but to date only genetic absence of alpha-l antiprotease has been definitely associated with COPD. An alternative, and not mutually exclusive, hypothesis advanced by Hogg and colleagues is that a latent adenoviral infection results in accumulation of neutrophils secondary to enhanced release of chernotactic factors (particularly IL-8). The combination of latent adenoviral infection and ETS exposure results in a threshold of inflammation sufficient to result in COPD. Interestingly, adenoviral EIA is detected in approximately the same percentage of the normal population as smokers that eventually develop COPD. Although there is considerable in vitro and animal evidence to support this hypothesis, there are comparatively few studies supporting this hypothesis in humans.
  
  Sputum examination and bronchoalveolar lavage (BAL) has greatly enhanced our understanding of lower respiratory tract processes. Sputum examination has the major disadvantage of frequent upper airway contamination and requiring evaluation by experienced personnel. BAL has the disadvantages of being invasive, inducing lower respiratory tract inflammation making it unsuitable for short-term observational studies, causing temporary worsening of hypoxia making it unsuitable for studying severely ill but non-intubated subjects, and high cost.
  
  The new technique of exhaled breath condensate (EBC) has several advantages over sputum and BAL. To perform EBC, a subject breathes normally into a non-occlusive tube. The subject’s exhaled breath that is saturated with water is condensed by cold and collected. EBC is noninvasive with little to no patient discomfort, can be performed in the severely ill, can be used for longitudinal sampling, and is much cheaper than BAL. The condensed sample contains non-volatile substances such as proteins, DNA, and RNA from lower airways that can be transported in the form of aerosols in exhaled air that can then be studied.
  
  
  
  
  
• Paul Switzer, Ph.D. 
  Determining the risk to public health for any environmental pollutant requires information on the concentration level and the duration of the population's exposure to that pollutant. The goal of this research is to measure, understand, and predict the exposure of the population to secondhand smoke, with emphasis on exposure at home, in motor vehicles, and at outdoor locations where smokers are present. Very few accurate measurements exist of the pollutant levels generated by environmental tobacco smoke (ETS) in these locations, despite widespread public health concerns about secondhand smoke. Secondhand smoke, or ETS, consists of more than 3,000 pollutants, both gases and particles. The levels of exposure revealed by these measurements are critical for evaluating the health risks of the population, and it is impossible to assess health risks without knowing the exposures. The exact level of exposure people receive from toxic pollutants due to smoking - ranging from a negligible exposure of only a few molecules to a massive exposure of thousands micrograms per cubic meter - is critical for determining the risk to human health, and these levels can be obtained only from careful measurements. Our research was the first to show that passengers in an automobile can be exposed to particulate matter concentrations as high as 2,000 mg/m3, and our research played a role in California's decision to restrict smoking throughout the State in public locations. This research also is relevant to other transportation micro environments, such as airplane passenger compartments. Our research builds upon our earlier measurements, following the indirect approach of exposure assessment, and it uses special models to generalize our findings to other cases and populations. Few measurements of the levels of exposure to ETS have been made in the home, and virtually no measurements exist on the levels of exposure people experience near smokers outdoors, Our research utilizes our extensive experience with state-of-the-art methods to make accurate measurements of air pollutants, including carbon monoxide (CO), particle mass concentrations (PM2.5 and PM10), particle sizes and size distributions, particulate polycyclic aromatic hydrocarbons (PAH), and volatile organic compounds (VOCs) such as benzene and xylenes, all of which are contained in tobacco smoke. This research proposes to: (a) determine quantitatively the levels of exposure that people receive to ETS; (b) quantify the microenvironments contributing the largest share of exposure in the population; (c) characterize the factors affecting ETS exposures in important microenvironments such as the home; (d) make new measurements of ETS pollutant concentrations in motor vehicles and in outdoor locations where smokers are present, (e) develop mathematical models to generalize these indoor findings to other cases and situations, (f) use our data to make risk assessments for protecting public health, and (g) combine the information into a new mathematical model to predict the effect of public health programs for reducing the population's exposure to tobacco combustion products. In addition to providing scientific measurement data needed to protect public health, our research provides information for preventive health - data that will be useful for reducing the exposure of the population to secondhand smoke.