The ultraviolet (UV) component of sunlight has been identified as a major etiological factor in the development of non-melanoma skin cancer (NMSC). Upregulation of Activator Protein-1 (AP-1) and Cyclooxygenase-2 (COX-2) have clearly demonstrated a functional role in skin tumor promotion. The goal of this work was to contribute to the growing knowledge of UVA and UVB induced signaling events leading to increases in AP-1 and COX-2. We show that UVA induces COX-2 expression in the human keratinocyte cell line, HaCaT through a post-transcriptional mechanism involving the 3 ' untranslated region (3'UTR). Use of a pharmacological inhibitor of p38 MAPK, SB202190, decreased UVA-induced COX-2 steady-state mRNA and protein levels. The stability of COX-2 mRNA is increased in UVA-irradiated cells and dependent upon p38 MAPK activity. We further explored the role of UVA-induced p38 MAPK activity in apoptosis in both HaCaT cells and primary keratinocytes. Dramatic increases in apoptosis were observed in UVA-irradiated cells treated with SB202190 or through the use of a dominant-negative construct. UVA induced expression of Bcl-X L with abrogation of expression using SB202190. Overexpression of Bcl-X L prevented PARP (Poly ADP-ribose Polymerase) cleavage induced by the combination of UVA and p38 MAPK inhibition. We further demonstrated that UVA enhanced the stability of Bcl-XL mRNA through increases in p38 MAPK activity mediated through the 3' UTR. p38 MAPK and Bcl-XL expression play critical roles in the survival of UVA-irradiated keratinocytes. Previous investigations from the laboratory identified p38 MAPK and PI3-Kinase as the major mediators of UVB-induced AP-1 and COX-2 in the HaCaT cell line. To further validate p38 MAPK and PI3-Kinase as potential molecular targets we investigated whether an acute UVB dose activated the p38 MAPK and PI3-Kinase pathways in vivo. We observed rapid increases in both p38 MAPK and PI3-Kinase signaling in mouse epidermis. Activation of these pathways resulted in the phosphorylation of cyclic AMP response element binding protein (CREB). Topical treatment with SB202190 or LY294002 (a specific inhibitor of PI3-Kinase) significantly decreased UVB-induced COX-2 expression and AP-1 activation in vivo. Our data suggest that p38 MAPK and PI3-Kinase may serve as significant molecular targets for the chemoprevention of UVB-induced NMSC.

Our laboratory had previously developed an in vitro tumor progression model which demonstrated that elevation of Reactive Oxygen Species (ROS) played a functional role in the development and maintenance of malignantly transformed mouse skin keratinocytes. The elevation of ROS levels were attributed to the repression of the anti-oxidant enzyme catalase. We hypothesized that repression of catalase, a potential tumor suppressor gene, led to the elevation of hydrogen peroxide which functions as a secondary messenger to active mitogenic signaling in the malignant cells. The first goal of these studies was to further characterize the repression of catalase in vivo. Using tumor samples generated by the mouse skin chemical carcinogenesis protocol, we determined that papillomas expressed higher levels of catalase protein and message than carcinomas. These results recapitulated the observations we made in the in vitro tumor progression model. The next goal was to determine the mechanism(s) behind the repression of catalase. Nuclear run-on analysis showed that catalase repression in the malignantly transformed cells was dependent on transcription. Results from luciferase reporter assays indicated that malignant cells have lower catalase promoter activities than benign papilloma cells, in part through the Wilm's tumor suppressor (WT1) binding site within the proximal promoter region. We concluded that WT1 element acts as a transcriptional repressor of catalase in this tumor progression model. The second part of this dissertation is focused on the role of the Rac1 signaling in tumor progression. Rac1 has been shown to activate NADPH oxidase to produce superoxide, potentially contributing to the elevation of ROS. We found that conditional expression of a dominant negative Rac1 was able to decrease multiple markers of malignancy including: growth, migration and invasion potential. In addition, these phenotypic changes were accompanied by a decrease in mitogenic signaling. Furthermore, we showed that inhibition of Rac1 signaling could reduce tumor growth in vivo. However, we were unable to show any decrease in intracellular levels of ROS. Based on these results, we concluded that Rac1 signaling plays a key role in mouse skin tumor progression through a ROS independent manner.

In light of the importance of preventive measures to effectively reduce the mortality rate associated with colon cancer, and the importance of adherence to treatment in the study of cancer prevention, the aim of this study was to identify variables that explain adherence to treatment in a long term, colon cancer prevention clinical trial. The Wheat Bran Fiber study is a double-blinded, placebo-controlled trial evaluating the efficacy of daily wheat bran fiber supplementation on polyp recurrence in 1,400 randomized persons with adenomatous colon polyp history. Adherence was defined as consumption of the cereal regimen during the specific evaluation period. It was evaluated through the count of returned unopened cereal boxes, and self-monitoring. Data were collected prospectively and discretely during clinic visits that took place approximately every three months. The Life-Style Questionnaire was constructed as a theory driven instrument with three major components: past adherence behavior, social network and self-motivation. I was given a baseline to 910 participants. The evaluation of potential predictors of attrition was conducted using survival analysis techniques. Specifically, a Weibull distribution was used to model the data. Demographic characteristics, physiological changes, and psychological variables constituted the explanatory variables of the model. The two most important predictors of attrition in this study, for both males and females, were exposure to high fiber and experiencing side effects, whether or not study related. Other significant predictors were self-motivation and adherence at baseline for males, and self-motivation, smoking history, and study expectations for females. One of the most interesting findings is the difference in risk between males and females. The higher hazard for females when toxicities were present suggests that women have more difficulties managing side effects. Also, smoking history was not a significant predictor of the hazard for males. Although the reason of such difference is unknown, it can be speculated that the motivations for and the circumstances under which males and females smoke might be completely different. Overall, these results confirm the multifactorial determination of attrition as a time-to-event phenomenon.

Protein tyrosine kinases (PTKs) mediate important signaling events associated with cellular growth, differentiation, and mitogenesis. The p60c-src protein is the first described cellular protein tyrosine kinase. Human p60c-src PTK has been implicated in the development of colon and breast cancer, and leukemia. However, the exact physiological role of p60c-src PTK or its physiological target proteins are not well known, and the mechanism by which the p60c-src PTK activity is regulated is not completely understood. Peptide substrates can be used to determine the substrate specificity and kinetic parameters, and therefore to provide important information for understanding of the physiological role and mechanism of action of this enzyme. Peptide substrates can also be used to develop pseudosubstrate-based peptide inhibitors for p60c-src PTK. Combinatorial peptide library methods have proven to be very powerful in identifying ligands for receptors and in discovering peptide substrates for protein kinases. In this dissertation, a "one-bead one-compound" combinatorial peptide library method was applied to identify peptide substrates for p60c-src PTK, the structure-activity relationship of the identified peptide substrates was studied, and the pseudosubstrate-based peptide inhibitors for p60c-src PTK were developed. Using the "one-bead one-compound" combinatorial peptide library method, a novel peptide, YIYGSFK, was identified as an efficient substrate for p60c-src PTK. The structure-activity relationship study was performed on over 70 analogs of YIYGSFK. It was determined that -Ile-Tyr- were the two critical residues required for activity. Based on this dipeptide motif a secondary library was synthesized (XIYXXXX, wherein X = all 19 eukaryotic amino acids except cysteine, I = isoleucine, Y = tyrosine) and screened with p60c-src PTK. One of the identified peptides, GIYWHHY, was found to be more efficient for p60c-src PTK than the parental compound, YIYGSFK. Several potent psedosubstrate based inhibitors were developed using GIYWHHY as a template. Some of the more potent inhibitors have branched structure indicating the enzyme active site can accommodate more than a linear motif. These data demonstrate that the "one-bead one-compound" combinatorial library method is a powerful tool to discover novel peptide substrates, and to develop pseudosubstrate-based peptide inhibitors for PTKs.

Microscopic fluorescence imaging of thick biological tissue has been successfully demonstrated with a fiber-based, slit-scanning, confocal microscope. The system developed under this research consists of an illumination arm, a fiber-optic imaging system, and a detection arm. The illumination arm is an anamorphic optical system that converts a circular, laser beam into a cylindrical beam forming a line image at the proximal face of the fiber-optic relay. This relay system is comprised of a fiber-optic imaging bundle, a miniature objective lens, and a miniature hydraulic positioning mechanism. It delivers illumination to a remote sample and simultaneously collects the fluorescence from the sample. The miniature objective lens and positioning mechanism were specially designed and fabricated for this system, allowing for high resolution imaging and optical sectioning in-vivo. The detection arm relays the fluorescence image at the proximal face of the fiber-optic relay with magnification onto a two-dimensional CCD. Characterization of the system has demonstrated a lateral resolution of three microns. The axial resolution when imaging a point object is 10 microns. When imaging a planar object, the axial resolution is 25 microns. Images are acquired at a rate of 2-4 frames per second and the imaging performance has been evaluated with different biological models including animal peritoneal tissue and human prostate tissue in-vitro. In-vivo images of human skin and rat peritoneum have also been acquired to demonstrate that patient motion does not adversely affect the performance of the system. These in-vitro and in vivo images demonstrate the capability of the system to resolve cell nuclear morphology, to visualize cell density and organization, and to image at selected depths below the tissue surface.

Human skin is continually subjected to UV-irradiation with p53 playing a pivotal role in repair of DNA damage and apoptosis. Mutations in p53 and p53 overexpression occur early in UV-induced skin carcinogenesis. We studied squamous cell carcinomas (SCC), actinic keratoses (AK), samples adjacent to AK (sun-damaged), and normal skin for p53 mutation, p53 immunostaining, apoptosis, and proliferation. The frequency of p53 mutation, p53 overexpression, and the number of apoptotic cells increased from normal skin, to sun-damaged skin, to AK, while AK and SCC had similar frequencies. Cell proliferation was significantly increased in the progression from normal skin to SCC. These data imply that apoptosis in samples with a high frequency of p53 mutation may not be p53-dependent. There may be two mechanisms for apoptosis. One is in response to DNA damage and the other in response to increased proliferation. It would be of great benefit to reduce the incidence of SCC and its precursor lesion, AK, through cancer chemoprevention. We applied surrogate endpoint biomarkers (SEBs) to skin cancer chemoprevention trials in subjects with multiple AK lesions. The primary SEB in these studies was reduction in the number of premalignant AK lesions, while secondary SEBs included p53 mutation frequency and exon distribution, p53 protein expression, and PCNA. An RXR-specific retinoid analog, targretin, demonstrated no significant reductions in SEBs, although there was a trend toward reductions in the number of AKs, PCNA, and p53 expression. This was a small study lacking power to detect a significant change. The ODC inhibitor, DFMO on the other hand, demonstrated significant reductions in the number of AKs, p53 expression, and although there was no reduction in the frequency of p53 mutations, there was a shift in exon distribution. Inhibition of polyamine synthesis in skin was determined in the DFMO study as a measure of drug effect. We conclude that measurement of SEBs in skin cancer chemoprevention studies is feasible, but it is imperative to choose SEBs that are appropriate to the chemopreventive agent and that have the potential for modulation. Sample size must have sufficient statistical power to detect a response to a chemopreventive agent.

Our lab has shown that the bile salt found in the highest concentration in human fecal water, sodium deoxycholate, induces apoptosis in several cell types including Jurkat cells as well as human colonic epithelial cells. We have also found that cells within the normal appearing flat mucosa of patients with a history of colon cancer are relatively resistant to apoptosis induced by NaDOC. The current studies test the hypothesis that sodium deoxycholate induces multiple stress response pathway s that protect against apoptosis. I have tested this hypothesis by developing and analyzing cell lines that are resistant to sodium deoxycholate-induced apoptosis and focusing on two stress-response proteins known to be activated by sodium deoxycholate, poly(ADP-ribose) polymerase (PARP) and the redo-sensitive transcription factor nuclear factor-kappa B (NF-κB). I found that PARP is protective against NaDOC-induced apoptosis, and by independently inhibiting the individual subunits of NF-κB, I found that the p65 subunit is protective, while the p50 subunit is not. Development and subsequent characterization of the NaDOC-resistant HCT-116 cell lines identified several proteins that may be responsible for the development of apoptosis resistance. These proteins will be further tested in future studies.

As the importance of developing anticancer agents specific to tumor cells grows, a need has arisen for new and better targets. It has been determined that, in addition to tumorigenic proteins and enzymes, DNA itself can be used to direct antitumor therapeutic agents. In recent years, the G-quadruplex DNA secondary structure has become of interest in terms of its use as a promising target in tumors. G-quadruplex DNA structures have been proposed to form in the promoter regions of a number of genes involved in cellular proliferation and tumorigenesis, including the proto-oncogene c-myc and many others. In this dissertation, the roles of a chair-type G-quadruplex structure in c-myc regulation and dysregulation, and its use as a target for antitumor agents, are investigated. It was found that such a structure very likely plays the part of a repressor of c-myc transcription, as its absence results in significant upregulation of this gene. Similarly, stabilization of this G-quadruplex with small molecules results in a reduction in c-myc expression, an activity lost when this structure can no longer form. It appears that this structure acts as a barrier to transcription factor binding, and Sp1 or an Sp1-like protein(s) may be the critical factor that is not permitted to bind when the quadruplex is present. Disruption of the G-quadruplex in the c-myc promoter, and subsequent upregulation of the gene, may play a role in the development of human tumors, possibly in the later stages of tumorigenesis, as mutations which abrogate quadruplex formation are found in patient tumor specimens and cultured tumor cells. Finally, a means of stabilizing the c-myc quadruplex with small molecules is developed using rational structure-based drug design, in order to take advantage of this repressor structure in a therapeutic context. Thus, there is elucidated a novel means of gene regulation heretofore unexplored, one that also represents a new frontier in anticancer drug design.

Human carcinoembryonic antigen (CEA) and the related CEA family member nonspecific cross-reacting antigen (NCA), function in vitro as intercellular adhesion molecules. Changes in the expression pattern of CEA and NCA in a wide variety of cancerous tissues has raised the possibility that they could contribute to neoplastic and malignant transformation by influencing tissue architecture and differentiation. This study revisits the expression pattern of CEA and NCA in colorectal tumorigenesis by emphasizing the accurate quantitation of cell-surface levels in the context of tissue architecture. FACSRTM analysis of highly purified tumor versus normal colonic epithelial cell suspensions, revealed dramatically higher levels of cell-surface associated CEA and NCA in tumor cells that was inversely correlated with histologic differentiation. Furthermore, a marked tendency towards delocalized expression and a multilayered tissue architecture was noted in less differentiated tumors. Increased levels of call-surface CEA and/or NCA (CEA/NCA) in colonocytes capable of differentiation was directly tested for effects on tissue architecture and differentiation with established in vitro assays and a novel in vivo colonic tissue architecture assay developed and presented as part of this thesis. The results demonstrate that overexpression of CEA/NCA on the surface of colonocytes can prevent cellular polarization, disrupt colonic tissue architecture and block differentiation both in vitro and in vivo.

DNA methylation is a postreplicative covalent modification of the DNA which is catalysed by the DNA methyltransferase enzyme. DNA methylation plays an important role in controlling the gene expression profile of mammalian cells. The hypothesis presented in this thesis is that the expression of the DNA methyltransferase gene is upregulated by cellular oncogenic pathways, and that this induction of MeTase activity results in DNA hypermethylation and plays a causal role in cellular transformation. Novel DNA methyltransferase inhibitors may inhibit the excessive activity of DNA methyltransferase in cancer cells and induce the original cellular genetic program. These inhibitors may also be used to turn on alternative gene expression programs. Therefore specific DNA methyltransferase antagonists might provide us with therapeutics directed at a nodal point in the regulation of genetic information.