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Introduction To facilitate review of this three-year competitive renewal proposal, I will summarize our past accomplishments, hypothesis, specific aims, and proposed future studies, in section A. Section B includes the budget, bibliography of the personnel, resources of the laboratory and collaborative efforts of the principal investigator. Section C will provide a detailed description and explanation of the methods to be used for the proposed study, as well as the references cited. Section D will list publications supported by the previous three-year grant period from the Philip Morris Co., Inc. Representative publications will be included in the Appendix.

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Table of Contents Introduction p.3 Section A: Summary of proposed studies I.Introduction p.4-7 II. Previous studies and results A. Studies with leukemia cells p.8 B. Prostate cancer cell studies p.8 C. Lung cancer cell studies p.9 D. Studies relating stress and atherogenesis p.10 III. Proposed studies p.11-13 Section B Detailed budget for initial budget period p.14 Budget for entire proposed budget period p.15 Budget justification p.16-17 Biographical sketch-Prirtcipal Investigator p.18-21 Other Biographical sketches p.22-28 Other support p.29-30 Resources and environment p.31 Section C: Detailed Research Plan I. Specific Aims p.32 II. Background and Significance p.32-36 A. Diet and modification of cancer risks B. Cell cycle control C. Apoptosis and carcinogenesis D. DNA repair III. Research Methods .36-42 A. Cell-biological assays B. Biochemical methods C. Molecular methods IV. Literature Cited .43-49 V. Human Subjects p.50 VI. Vertebrate Animals p.50 Section D Publications supported by an unrestricted grant from Philip Morris, Inc., 95-98 p.51-54 A. Leukemia cell studies B. Studies with prostate cancer cells C. Lung cancer cell studies D. Studies relating stress and atherogenesis Appendix Publications ~

Section A: Summary of Previous Accomplishments and Proposed Future Studies I. Introduction Despite the progress made in cancer diagnosis, methods for early detection, improved treatment modalities, and cellular, biochemical and molecular characterization of many forms of malignancy, and significant gains in the treatment for a few specific types of cancer [11, the overall mortality rates for most cancers and the total cancer incidence have remained disproportionately high and continue to rise in westernized countries and cultures.[2]. Statistics indicates that more than 30% of people will be diagnosed with cancer during their lifetime and approximately 25% of total deaths may be ascribed to cancer. A particularly significant increase has been noted in new cases of breast cancer in females, prostate cancer in males, and lung ca .er in both sexes, in recent decade [3]. Although a multitude of factors including environment, hormones, lifestyle and inheritance has been linked to the disease, a unifying theory of carcinogenesis has remained elusive. What is clear, however, is that cancer is fundamentally a genetic disease at the cellular level, whose evolution from normal cells involve multiple genetic events, for which there are a multiplicity of steps leading to these events; the cumulative result is a disruption'of the normal regulatory pattern characterizing normal cells. Phenotypically therefore, cancer is characterized by uncontrolled cellular growth and proliferation, a reduced capacity or loss of fidelity of DNA repair, and lack or dysfunction of programmed cell death, also referred to as apoptosis (Figure 1). Figure 1. Events and markers in relation to exposure and disease 4

r. ~ e o e = - F y 4 J Genetic events leading to cancer may be triggered by exogenous exposures, endogenous exposures, to potential carcinogens, or enzymatic errors in macromolecular syntheses. For most cancers, however, the establishment of a cancerous state cannot be explained by a single gene defect or an episodic exposure to adverse environmental challenges, but is more likely to be caused by the interactions of multiple genomic and environmental factors, with distinct as well as overlapping biological consequences [1-5]. This complex, multifactorial etiology implies that there will be etiologic heterogeneity among cancer cases in the general population [2,6], suggesting that the tracking of the cause for a given cancer type is subject to confounding and making optimization of cancer prevention, detection and treatment a formidable if not an unachievable task. Because of the recognition that we lack ability to easily trace the etiology or eradicate invasive cancers, a new scientific view and perspective has emerged and gained prominence in recent years for.4owering the cancer incidence and mortality, namely, to intervene 5

cancer at earlier disease stages. The consensus among experts is that such intervention can best be done by naturally occurring substances in the diet [2,5]. Dietary factors may contribute to as much as a third of potentially preventable cancers and the long-term preventive effect of plant-based diets on tumorigenesis is supported by extensive epidemiological and laboratory studies. Fruits and vegetables, herbs and spices and specific food ingredients have been reported to contain antimutagenic and anticarcinogenic potentials [2,7,8]. However, their broad use to prevent, inhibit, or even reverse carcinogenesis is limited by the fact that in most cases, the manner by which diet/nutrition and specific dietary component(s) function as chemopreventive agents has not been systematically investigated and rigorously tested. The long-term interest and objective of this laboratory has been to elucidate the mechanism of action of microchemicals and specific dietary components. For the past several years, we have focussed on investigating the effects of retinoids, e.g., retinoic acid RA and fenretinide 4HPR, antioxidants, e.g., resveratrol, and vitamins, e.g., vitamin D, on a number of cultured cancer cells. Our hypothesis is that these agents may act in common as pleiotropic cellular regulators to selectively effect multiple changes in target cancer cells, the net result of which is that cancerous cells become non-carcinogenic. Possible proposed mechanisms include: (i) antimitogenic effects in which these agents act by blocking G1/S transition of the cell cycle, (ii) apoptosis-activating effect that results from the ability of these agents to regulate mitochondrial structure/function and consequentially the expression of the bcl-2 family of proteins and activation of caspases, and (iii) modulation of DNA repair capacity and/or activity, mediated by changes in p53, poly(ADP- ribose) polymerase (PARP), and the double-stranded DNA-dependent protein kinase (DNA-PK) (Figure 2). To validate the multiplicity of their actions, we have resorted to studying, in detail, the effects of representative agents on model tissue culture systems and on expression of several key regulatory protein molecules mentioned above. Key findings of these studies are presented below. N Cn O 9 O J 6 w

Salary will be adjusted upwards by 4% in years 2 and 3 to cover the cost of inflation. The same percentage of increase will apply to the supply and miscellaneous categories.

The sequence of experiments regarding the above objectives is summarized below and in Figure 3. Specific Aims One: Control of G1/S Two: Control of Three: Control of transition a o tosis DNA re air Target Cyclin D1 Bcl-2 family P53 genes/proteins Cdk 4,6 Cytochrome c PARP proposed for analysis pRB, CKIs (p16, p2l Caspases DNA-PK and p27) Proposed Experiments For each Target Gene/Protein Objective A roach Si ni5cance Control of expression • Western blot analysis • Whether protein level changes of protein level • Metabolic labeling and • Whether protein synthesis immunoprecipitation accounts for change in protein levels Post-translational • Phosphorylation and • Determine importance of covalent modification of dephosphorylation of modification proteins protein • Specific processing of • Evaluate role of proteolysis roteins Control of RNA level • Northern and dot blot • Distinguish transcriptional from changes analysis post-transcriptional control • Actinomycin D and chase • Nuclei run-on experiments • Nuclease sensitivity assay • Evaluate importance of DNA • Transfection experiments regions important for control of • Gel mobility shift assay gene expression • Affinity labeling of nucleic • Demonstrate existence of acid binding factors chemopreventive agent responsive rotein re ulato factors Role of ceramide • Fumonisin B to block • Importance of ceramide ceramide synthesis biosynthesis in signalling of a o tosis Role of caspases • Inhibitors, e.g., DEVD for • Distinguish involvement of CPP32, YVAD for ICE s ecific cas ases 12

- .-s-.F-a.' . Figure 3. Summary of Proposed Studies ' e.g.4-1-IPR, ~ ~ resveratrol ' ,-4Chemo-agent -, Blockins GI/S DNA Repair 1 •e, G 1 IN S DNA damage pR-B -lo pRB. ~ un(der) phosphorylated i focm ~ E2F Cyclin D/CDK4~ hypelphosphorylated form p53*,d 0 p53 PARPA i L --* Apoptosis Apoptosis Survival sienai Death sieual bcl-2 -lo bcI-2* phosphorylated form ba or bcl-2 bc12/bax (complex) or Protease activity PARP + ed substrate (PARP, actin, chromatin) Y DNA repair can not repair ~ ~ ~ L ------------------jj~ 13 phosphorylated wj)dtype phosphoryl fo I . form Apoptosis