The Ki-ras oncogene and the p53 tumor suppressor gene are mutated in a high percentage of human colon cancers. The mutation status of these two genes were assessed in colon adenocarcinomas obtained from azoxymethane (AOM)- and dimethylhydrazine (DMH)-treated rats, widely used experimental models of human colon carcinogenesis. The status of p53 mutations was examined using polymerase chain reaction (PCR) amplification of these sequences. In so doing, it was discovered that the rat p53 gene is structurally distinct from the human p53 gene, since it is missing one intron between exons 6 and 7. Further analysis using single stranded DNA conformational polymorphism (SSCP) analysis and direct DNA sequencing of the highly conserved regions of rat exons 5-7, highly mutated in human colon cancers, revealed no p53 mutations in any of these regions. Mutations at codon 12 of the Ki-ras gene were also characterized. Mutation frequency was approximately 60% which is close to the frequency of Ki-ras mutations found in human cancers. Since Ki-ras mutations have been shown to occur early in human colon carcinogenesis, while p53 mutations are thought to occur in late stage tumors, these data suggest this model may be a good for early, but not late, events of human colon carcinogenesis. It was also found that the non-steroidal anti-inflammatory drug (NSAID), sulindac sulfone, could reduce Ki-ras mutation frequency in this model. NSAIDs are agents being studied as a possible chemopreventive agent in both human colon cancers and the AOM model. Previous studies have found that the NSAIDs, sulindac and its sulfone metabolite, decrease the frequency of colon tumors in the AOM model. In this study, sulindac and sulindac sulfone specifically induced apoptosis in normal rat embryo cells (RECs) transfected with either an EJ-ras or activated myc and ras but not in non-transfected RECs. These data suggest that sulindac and sulindac sulfone may be reducing tumor frequency and Ki-ras frequency by inducing apoptosis in those cells with specific mutations that give them carcinogenic potential, possibly through non-prostaglandin dependent pathways.

The mouse skin model that has been used to study skin carcinogenesis can be divided into three stages: initiation, promotion, and progression. One genetic change observed during tumor promotion and malignant progression is increased transactivation of the transcription factor AP-1. AP-1 consists of Jun (c-Jun, Jun B, Jun D) and Fos (c-Fos, Fos B, Fra-1, Fra-2) proteins that form Jun:Jun homodimers or Jun:Fos heterodimers. AP-1 binds to a consensus cis-promoter element, the TRE, and transcriptionally regulate a number of genes with various biological functions. By studying the benign mouse keratinocyte cells, 308, and its malignant variant, 10Gy5, it has been shown that 10Gy5 cells have elevated AP-1 activity compared to 308 cells. Reduced AP-1 transactivation in 10Gy5 cells has been correlated with suppression of its malignant phenotype. This research examined the differential AP-1 transactivation in benign 308 and malignant 10Gy5 cells. By examing mechanisms of AP-1 regulation in the two cell lines, differences were observed with post-translational modifications of AP-1. There were differences in phosphorylation of one of the AP-1 family members, Jun B. In addition, AP-1 proteins in 10Gy5 cells appear to be in a fully reduced state, unlike AP-1 proteins in 308 cells. A third difference that was observed was in Jun B steady state protein levels, with decreased Jun B protein in malignant 10Gy5 compared to benign 308 cells. Reduced Jun B protein in 10Gy5 cells was the result of decreased Jun B protein synthesis. Jun B protein may inhibit AP-1 transactivation and cell proliferation. Experiments were performed to determine whether Jun B protein could modulate AP-1 transactivation, cell growth, and tumor formation in 308 and 10Gy5 cells. Altering Jun B protein levels in these keratinocytes affected AP-1 transactivation. Overexpression of Jun B protein in malignant 10Gy5 cells corresponded to an inhibition of cell growth and tumor development. However, overexpression of Jun B protein in 10Gy5 cells was not sufficient to reverse the malignancy, indicating that additional genetic changes are involved in malignant conversion of these keratinocytes. The results of this research suggest that Jun B protein levels may be important during malignant progression of mouse skin.

Bile acids have been linked to the etiology of colon cancer in various studies for over twenty-five years. However, the mechanism by which bile acids act in colon carcinogenesis is not known. By using an assay that can detect induction of the gadd153 stress response to DNA damage, I found that bile acids activate expression of gadd153 promoter. This observation implies that bile acids cause DNA damage. I then hypothesized that exposure of cells to bile acids produces reactive oxygen species which damage DNA. However, experiments to block gadd153 induction by bile acids using antioxidants, and to measure 8-OH-dG induced by bile acids were inconclusive. I also used p53 mutant cell lines to show that bile acid induction of apoptosis is p53-independent. In addition, my experiments showed that bile acid increased expression of gadd153 protein in HT-29 cells, and that gadd153 protein was constitutively overexpressed in HCT-116 cells with or without bile acid treatment. Thus, bile acid action in colon carcinogenesis may involve induction of DNA damage, induction of gadd153 as a protective stress response, and then if the damage is beyond repair, p53-independent apoptosis.

Ionizing radiation is a physical agent that is tumorigenic in all exposed tissues. These radiation-induced secondary neoplasms tend to be more aggressive and carry a poor prognosis. Our knowledge of the molecular mechanisms of ionizing radiation carcinogenesis is not as advanced as compared to chemical carcinogenesis. We have used repeated exposure to low LET radiation in the mouse skin model to study the molecular mechanisms of ionizing radiation as a complete carcinogen and as a tumor progression agent. Shaved backs of CD-1 mice were treated with fractionated doses of beta-irradiation in a complete carcinogenesis experiment. A total of 27 carcinomas and sarcomas were seen. Cell lines were established from four sarcomas and one squamous cell carcinoma. Biochemical studies revealed that three sarcoma cell lines were derived from rhabdomyosarcornas. All four sarcoma cell lines had a p53 null phenotype. We screened cDNA expression libraries from three cell lines for dominant transforming activities. GAPDH was isolated as a candidate transforming gene in the squamous cell carcinoma cell line. Using a papilloma producing mouse keratinocyte cell line, we have shown that repeated doses of ionizing radiation are equally effective as a tumor progression agent when compared to N-methyl N'-nitro-N-nitrosoguanidine (MNNG). In this model, elevated reactive oxygen species levels were seen in both radiation and MNNG progressed cells. Elevated transcription factor transactivation as well as constitutive activation of Erk-1/2 and p38 MAP kinase activities were found to be potential mediators of the reactive oxygen species mediated mitogenic signaling in the progressed phenotype. Analyses of the anti-oxidant defense mechanisms showed that attenuation of catalase activity was a potentially important mechanism for the establishment of the pro-oxidant state. Forced re-expression of catalase in the malignant variants resulted in a reduction in transcription factor transactivation. Taken together, the results from experiments presented in this dissertation suggest that inactivation of gene products that maintain genomic stability, such as p53, may be an important step during neoplastic transformation with fractionated doses of ionizing radiation. Altered expression patterns of genes related to cell metabolism and oxidative stress can be functionally involved during the later stages of ionizing radiation-induced malignant transformation.

The major mediator of the 3-phosphoinositide signal transduction pathway is phosphatidylinositol (PtdIns) 3-kinase (Kapellar and Cantley, 1994). The study of the 3-phosphoinositide pathway has been facilitated by the existence of potent irreversible inhibitors of p110 PtdIns 3-kinase, such as wortmannin (WM) (Powis et. al, 1994). Anti-metabolites of the 3-phosphoinositides generated in this pathway, such as 1D-3-Deoxyphosphatidylinositol (3'Deoxy-PtdIns), are also useful tools. 3'Deoxy-PtdIns is an analog of PtdIns which, although it can be phosphorylated at the 4 or 4 and 5 positions of its inositol ring, is an anti-metabolite of 3-phosphoinositide signaling molecules (Kozikowski et al., 1995). In this study WM and 3'Deoxy-PtdIns were used to determine whether the 3-phosphoinositide pathway was a useful target for the development of a anti-cancer therapy. We found that WM and 3'Deoxy-PtdIns both inhibited the growth of murine C3H and human MCF-7 mammary tumors in vivo, however WM did not inhibit the growth of human UACC2150 tumor. The ability of WM to inhibit C3H tumor growth was not related to inhibition of tumor total PtdIns 3-kinase activity. The existence of the WM-insensitive PtdIns 3-kinase activity observed in these tumors was confirmed in C3H and MCF-7 cell culture lysates, solid tumors and tissue homogenates. In addition to being resistant to inhibition by WM, MCF-7 cell lysate total PtdIns 3-kinase activity was also resistant to five known p110 PtdIns 3-kinase inhibitors. Human normal colon mucosa, colon tumors and placenta also contained WM-insensitive populations of PtdIns 3-kinase activity. Human placental homogenate was chosen as the source for the purification of WM-insensitive Ptdlns 3-kinase because it contained a profile of PtdIns 3-kinase activity which was similar to that of MCF-7 cells. Purification of WM-insensitive PtdIns 3-kinase from human placenta did not result in the identification of PtdIns 3-kinase related proteins. Although WM appeared to inhibit tumor growth by a non-PtdIns 3-kinase dependent mechanism, the results of this study confirmed that the 3-phosphoinositide signal transduction pathway was involved in the growth of mammary tumors. Because of its predominance in solid tumors and normal tissues, the WM-insensitive PtdIns 3-kinase, once identified, may be a suitable target for anti-cancer drug development.

Transforming growth factor-beta (TGF-β) is a multi-functional cytokine produced by many tumor cells that is known to potently inhibit immune cell functions. Secretion of TGF-β by malignant cells may therefore be a mechanism by which tumor cells escape destruction by tumor-specific T lymphocytes. If this is the case, prevention of TGF-β production by tumor cells could be expected to eliminate tumor-derived immunosuppression and enable the development of effective anti-tumor immune responses. To evaluate the role of tumor-derived TGF-β on tumor progression, production of this cytokine was interrupted by introducing a gene encoding antisense TGF-β1 into the EMT6 murine mammary tumor cell line using a retroviral vector (LasTGF-β1SN). EMT6 cells transduced with this vector (EMT6asTGF-β1) stably expressed the antisense gene and secreted 48% less TGF-β than did tumor cells transduced with the backbone vector alone (EMT6-Neo). Supernatant fluid recovered from tumor calls expressing the antisense TGF-β1 gene also exhibited a decreased capacity to suppress alloantigen-specific cytotoxic T cell responses in vitro. Furthermore, tumor growth in mice injected with EMT6asTGF-β1 tumor cells was inhibited compared to mice injected with control EMT6-Neo tumor cells. Immune cell involvement in the growth inhibition of the antisense TGF-β transduced tumors was suggested by the ability of EMT6asTGF-β tumors to grow unchecked in immunodeficient scid mice and by the finding that immunocompetent mice that rejected antisense TGF-β1-expressing tumors developed long term tumor immunity. These results demonstrate that expression of antisense TGF-β1 by transduced EMT6 cells decreases their tumorigenicity and that this approach of eliminating immune suppression is a potentially useful strategy to enhance antitumor responses.

Drug resistance continues to be one of the major obstacles to the successful treatment of hematopoietic cancers. Historically, most of the research being done in this area has been focused on reversing mechanisms of resistance which are known to be acquired by the tumor cell in vitro. Fewer studies have attempted to determine what intrinsic mechanisms allow these cells to resist the apoptosis inducing effects of cytotoxic drugs in an in vivo setting. It is now known that a tumor cell's interactions with its microenvironment can have a major influence on whether it lives or dies. The research presented in this dissertation shows that tumor cell interactions with the extracellular matrix component fibronectin influence cell survival following cytotoxic drug exposure, a phenomenon which has been termed Cell Adhesion Mediated Drug Resistance (CAM-DR). In the 8226 myeloma cell line, p130Cas is activated by Src following fibronectin (FN) adhesion, but this signaling pathway did not have an immediate effect on drug response. Similarly, members of the MAPK family, including ERK1/2 and p38, were found to be inactivated by FN adhesion but not to play a short-term role in suppressing drug induced apoptosis. Furthermore, experiments utilizing the K562 CML cell line demonstrated that cytoprotective signaling by the integrins is independent of AKT and BCR/ABL-associated signaling. By allowing the malignant cell to avoid cytotoxic drug induced apoptosis, the integrins may play a significant role in the generation of tumors which are refractory to chemotherapy. Pharmacological agents targeted at these adhesion molecules or at downstream signaling partners have the potential to potentiate the actions of anti-tumor agents and improve clinical responses.

The RNA expression of the tumor suppressor genes BRCA1 and maspin are frequently decreased or lost in sporadic breast cancer. We hypothesized that inactivation of these genes was due to aberrant epigenetic silencing at the level of gene transcription. In this study we show that aberrant cytosine methylation of the BRCA1 and maspin CpG island promoters is a common event in the inactivation of these genes in sporadic breast cancer cell lines. Furthermore, we show that the methylation-associated inactivation of BRCA1 occurs in 15-30% of sporadic breast cancer patient specimens and our data suggests that the methylation-associated inactivation of maspin occurs in ∼70% of advanced sporadic breast cancers. Additional studies indicate that the methylation associated inactivation of these genes is coincident with the repressive epigenetic events of histone hypoacetylation and chromatin condensation. These data suggest that aberrant cytosine methylation, histone hypoacetylation and chromatin condensation act together in the BRCA1 and maspin promoters to inactivate their transcription, thereby, contributing to the progression of sporadic breast cancer.

Previous work from our laboratory indicated that the bile salt, sodium deoxy-cholate (NaDOC), induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa, and that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Secondary bile acids are known promoters of colon cancer, but the mechanism by which they promote cancer is still largely unknown. We have shown that high physiologic concentrations (0.5 mM) of NaDOC activates the redox-sensitive transcription factor, NF-κB, and also causes the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of cells. These observations indicate that this bile salt induces oxidative stress within the cells. Since peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, L-NAME (N-nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, CuDIPSH, a superoxide dismutase mimetic compound, Trolox, a water-soluble analog of α-tocopherol, Melatonin, a fat and water soluble antioxidant, N-acetyl-cysteine, a GSH enhancer, U-74389G, a lazeroid that inhibits superoxide anion and free radical lipid peroxidation and U83836E, a lazeroid that is 100X more potent than trolox, alone, or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. We also investigated the effects of inhibitors of certain pathways known to generate ROS, mitochondrial complexes I and II of the electron transport chain and arachidonic acid metabolism, on bile salt-induced apoptosis. Both rotenone and TTFA, inhibitors of mitochondrial complex I and complex II respectively, protected HT-29 cells from NaDOC-induced apoptosis. The inhibitor of COX-1, Sulindac Sulfide, sensitized cells to NaDOC-induced apoptosis and so did the combination of the COX-2 and LOX inhibitors, NS-398 and Esculetin. These results suggest that nitric oxide and reactive oxygen species (ROS) may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that antioxidants may possess anti- cancer properties.

Tumor cells can be modified with cytokine genes such as the Interleukin-2 (IL-2) gene. The levels of IL-2 expressed are critical for successful treatment. We have tried to achieve higher levels of IL-2 than those currently available by conventional plasmids. Use of a transcriptional activator, e.g; the tat gene along with the HIV promoter driving the IL-2 gene, greatly increased IL-2 levels compared to widely used cytomegalovirus (CMV) driven plasmids. Control of the tat gene with an inducible promoter, i.e; the human HSP70B promoter, permitted control of gene expression. The inducibility of the HSP70B promoter by heat, γ-radiation and geldanamycin (a chemotherapeutic drug) allowed for a combinatorial approach to cancer treatment with hyperthermia, radiation therapy and chemotherapy. Also a brief heat treatment of 10 min at 42°C of target cells increased plasmid uptake, and higher levels of gene expression could be achieved. Another arm of immunotherapy is adoptive therapy with Tumor Infiltrating Lymphocytes (TILs). Insufficient numbers of tumor-specific T-cells limit the success of TIL therapy. An alternative approach to overcome this limitation is to transfer tumor-specific T cell receptor (TCR) into peripheral T-cells, redirecting their specificity to the tumor cell. To prove the feasibility of this technique, T-cell receptors were identified and cloned from hybridomas specific for the tumor cell line, MO5. A three domain single chain T-cell receptor was also constructed from the tumor-specific TCR genes to investigate the ability of a single chain T-cell receptor to activate T-cells. The CD3ζ chain was linked to the single chain to allow signal transduction upon antigen recognition by the TCR. The full length and the single chain TCR were cloned into a retroviral vector and transfected into mouse and human T cell lines. Cell surface expression of the chains were detected by flow cytometry. Functionality of the transfected TCR chains was assessed by IL-2 secretion on co-culture of the tumor cell line MO5 and the transfected T-cells. The two different approaches described here, i.e; higher levels of IL-2 for IL-2 gene therapy and specific redirection of T-cells can potentially greatly enhance the success rate of cancer treatment.