Malignant gliomas represent over 50% of the tumours which originate within the central nervous system. Current treatment modalities are in large part unsuccessful, and less than 10% of patients with high grade glioblastomas survive beyond 2 years. Understanding the cause of the rapid growth rate of malignant gliomas would be useful in devising new therapeutic strategies to treat this disorder. Previous work from this laboratory suggested that the enzyme protein kinase C (PKC) was a critical regulator of the hyperproliferative state of glioma cells. The results presented in this thesis demonstrate that (1) PKC activity was differentially increased in glioma cell lines when compared to other fast growing cell types of non-glial origin, (2) inhibitors of PKC (staurosporine, tamoxifen, CGP 41 251) block the proliferation rate of glioma cells at IC50 values that correspond to their respective IC50's for inhibition of PKC activity, (3) of the PKC isoforms, rat astrocytes and rat glioma cells contain $ alpha, beta, delta, varepsilon$ and $ zeta$, but not $ gamma$, (4) of the expressed isoforms, the activity and amounts of PKC$ alpha$ correlate with cellular growth and (5) an anti-sense oligonucleotide directed against PKC$ alpha$ decreases the proliferation rate of glioma cells. These observations suggest that aberrations in PKC signal transduction (particularly PKC$ alpha$) are important factors in the growth of glioma and have led to clinical trials utilizing PKC inhibitors as adjuncts in the therapy of patients harbouring these incurable neoplams.

CEA-related cell adhesion molecule 1 (CEACAM1), a member of the carcinoembryonic antigen (CEA) family, is downregulated in a variety of malignancies including colon, prostate, liver, breast and endometrial cancers. Previous studies have shown that expression of the CEACAM1 isoform containing the long cytoplasmic domain (aa 448--521 in mouse; CEACAM1-L) inhibited tumor growth in vivo, while the expression of the CEACAM1 isoform bearing the short cytoplasmic tail (aa 448--458 in mouse; CEACAM1-S) had no effect. Therefore, the long cytoplasmic domain contains determinants which are crucial for the growth inhibitory phenotype. We have demonstrated that sequence elements within the C-terminal region of CEACAM1-L are responsible for the inhibition of colonic tumor cell growth. Mutation of Tyr488, Va1518 and Lys519--521 averted CECAM1-L's tumor suppressive activity. Furthermore, the role of CEACAM1's adhesive properties in the tumor inhibition phenotype was also investigated. Truncation of the N-terminal domain of CEACAM1 abrogated CEACAM1's ability to sustain intracellular adhesion but had no effect on the tumor inhibitory function. Hence, while the adhesion domain is not implicated in CEACAM1's tumor inhibitory activity, the C-terminal region of the long cytoplasmic domain plays an important role.

Since lymph node stromal cells remain largely uncharacterized with respect to cell surface markers and function, their role in regulating the growth and invasion of disseminated cancer cells, including breast carcinoma has, to date, been virtually unexplored. In the present study, we asked whether peripheral lymph node cells could modulate the growth of breast carcinoma cells and, thereby, contribute to the progression of the metastatic process. Primary cultures of rat peripheral lymph node stromal cells were obtained by limiting dilution and two sublines, STA4 and STB12, with breast carcinoma growth-promoting activities were isolated. Immunocytochemistry performed on these cells revealed that they express vimentin, S-100 and fibronectin, but neither cytokeratin nor von Willebrand factor indicating that they are stromal and dendritic in origin. Several functional studies were performed using media conditioned by STA4 and STB12 cells. (Abstract shortened by UMI.)

The use of tissue or tumor selective promoters in targeted gene therapy for cancer depends on strong and selective activity. Hexokinase type II (HK II) catalyzes the first committed step of glycolysis and is overexpressed in tumors, where it is no longer responsive to normal physiological inhibitors, e.g. glucagon. I show in a reporter gene assay activation of HK II in non-small cell lung carcinomas NCI-H661 and NCI-H460 at 61% and 40% of the activation observed with a constitutive promoter respectively, while it is only 0.9% in a number of different primary normal human bronchial epithelial cell lines (NHBEC). Similar results were observed in a variety of normal and tumor cells. Moreover, treatment of the transfectants with glucagon did not inhibit promoter/activation in the transformed H661 cells, while endogenous HK II in NHBEC is suppressed by glucagon, H460 and H661 cells infected with a recombinant adenovirus carrying a HK II/LacZ expression cassette, Ad HexLacZ, demonstrated beta-galactosidase activity, which correlated with the level of HK II promoter activation in these cells. Under similar conditions, no enzyme activity was observed in NHBEC. Cells were then infected with AdHexTk and treated with GCV. Our results demonstrate selectivity in toxicity with a 10--100 fold increase in IC 50 between lung cancer cells and NHBEC. There was also a 100-fold increase in IC50 in normal human mammary epithelial cells (NHMEC) relative to breast carcinoma cells MCF-7. This represents a novel use of the hexokinase type II as a selective promoter in cancer gene therapy. Other factors important in suicide gene therapy were explored. Pharmacological modulation of the bystander effect using 8-bromo-cAMP, observed in HSVTk/GCV suicide killing, was demonstrated to enhance killing efficacy by 50% when a small proportion of a target population was gene modified. The phenomenon of cellular resistance to HSVTk/GCV was examined and in a novel finding, we demonstrated dramatic int

5-Oxo-L-prolinase (5-OPase) is involved in the synthesis and metabolism of glutathione (GSH) in the gamma-glutamyl cycle. The present project is to investigate (1) the level and distribution of 5-OPase in human tissues, (2) the potential application of a 5-OPase modulator, L-2-oxothiazolidine-4-carboxylate (OTZ), in selective modulation of GSH in normal versus tumor cells in cancer chemotherapy, and (3) the role of 5-OPase in cellular GSH modulation. / First, Compared to rat tissues, 5-OPase activity was found to be generally low in human tissues and no specific distribution pattern was observed. A polyclonal rabbit anti-rat 5-OPase antibody was produced with high affinity and specificity and it was shown to crossreact with human 5-OPase. Immunohistochemistry analysis revealed that 5-OPase was localized in the structures that have typical absorptive or secretary function. By Western blot, normal human lung and stomach tissues showed significantly higher 5-OPase levels than their paired tumors, while the other tissues examined showed case-dependent patterns. / Second, OTZ, a 5-oxo-L-proline analog that is metabolized to cysteine by 5-OPase, was shown in vivo to increase the GSH level in normal bone marrow cells while paradoxically decreasing it in rat mammary tumors and sensitizing the tumors to the alkylating agent melphalan. When the human breast cancer cell line MCF7 was treated with OTZ in combination with melphalan, the cytotoxicity of melphalan was increased relative to that of melphalan alone, and this effect was reversed by the addition of glutamate or 5-oxo-L-proline. OTZ treatment decreased the GSH levels in MCF7 cells but increased the GSH levels in normal human breast MCF10A cells. Therefore, in tumor cells where glutamate is limited in GSH synthesis, OTZ increases melphalan toxicity by limiting glutamate production from 5-OPase for GSH synthesis. / Third, the expression of transfected 5-OPase in MCF7 cells decreased the cellular GSH level, sensitized the Cells to melphalan toxicity, and diminished the sensitizing effect of OTZ on melphalan toxicity; while exposure of both normal and cancerous human cells to GSH depleting agents led to an increased expression of 5-OPase both in vitro and in vivo. These results indicate a critical interaction between cellular GSH levels and 5-OPase activity that could be important in GSH modulation in therapeutic settings.

Human retinoid X receptor alpha (hRXRalpha) plays a critical role in DNA binding and transcriptional activity through its heterodimeric association with several members of the nuclear receptor superfamily, including the vitamin D receptor (VDR). Several cancer cell lines derived from many tissues have been shown to be resistant to the growth inhibitory action of 1,25-dihydroxyvitamin D3, (1,25(OH)2D3), the biologically active metabolite of vitamin D3. In the malignant ras-transformed human keratinocyte cell line, HPK1Aras, 10--100 fold higher concentrations of 1,25(OH)2D3 are required than the non-malignant normal human epidermal keratinocytes to achieve comparable inhibition of cell growth. Here we show that in ras-transformed keratinocytes, ser260 of hRXRalpha is phosphorylated through the Ras-Raf-MAP kinase cascade. This phosphorylation event results in the inhibition of vitamin D signaling via VDR/hRXRalpha heterodimers. Strategies to reverse this resistance include the use of the MAP kinase inhibitor, PD098059, and a non-phosphorylatable hRXRalpha mutant, ala260, which completely abolishes RXR phosphorylation and restores the function of both 1,25(OH)2D3 and a specific RXR ligand, LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl)ethenyl]-benzoic acid). In addition, we show that a vitamin D analog with low calcemic activity (EB1089) is more potent than 1,25(OH)2D3 in inhibiting cancer cell growth in this system. Targeted therapy with selective analogs such as EB1089, in combination with the inhibition of phosphorylation of the RXR, could play a critical role in the therapeutic strategies of cancer biology. In addition, we also demonstrate that resistance to 1,25(OH)2D 3 can be acquired through genetic alterations in the VDR, implying that both components of the VDR/RXR heterodimer are potential targets for the induction of cellular resistance to 1,25(OH)2D3 and present two distinct mechanisms through which tumour cells can escape the growt

Nuclear factor-erythroid 2 (NF-E2) is a basic leucine zipper (bZIP) transcription factor that recognizes the DNA binding site [(T/C)GCTGA(G/C)TCA (T/C)] that contains a central AP-1 motif (underlined), but is distinct from AP-1 factors (Jun, Fos) due to the requirement of residues outside of the AP-1 core. NF-E2 is a heterodimer comprised of a cap'n'collar factor (p45) and a small Maf protein (MafF, G or K). It has been shown that NF-E2 promotes the opening of chromatin throughout the beta-globin loci, promoting gene expression and erythroid differentiation. Also, the p45 subunit is restricted to hematopoietic progenitor, erythroid, megakaryocytic and mast cells while the small Mafs are ubiquitously expressed. / I have used the erythroleukemia cell lines, MEL and CB3, as model systems to study the role of NF-E2. MEL cells can be induced to differentiate, while CB3 cells, which do not express p45 due to a Friend leukemia virus insertion in one allele and the loss of the other, express only minimal levels of alpha- and beta-globin mRNA upon induction.

A subset of the leukaemic clone, the leukaemic stem cell (L-HSC), is responsible for the maintenance and propagation of a leukaemia. Most current therapies, however, do not target this population. In this thesis, I show that a determinant of disease phenotype is the frequency of the leukaemic stem cell within the leukaemic population. Moreover, the frequency of L-HSC in a leukaemia is predetermined by the inherent properties of the cell that is transformed and can be attributed to the ontogenic origin of the cell. Ideal therapies would specifically target mechanistic defects in leukaemic stem cells; however, the pathways that are involved and what oncogenic defects can be targeted for therapy remain to be discovered. As it is not known if all oncogenes can be targeted or what the determinants of oncogenic dependency are, I have developed a system to further elucidate this as described herein.

A key reason for malignant glioma recurrence after radical tumor resection is the failure to control individual invading tumor cells that have moved away from the primary tumor mass. The purpose of this study was to assess the role of ROCK (Rho-kinase), a signaling protein in the Rho GTPase pathway, in malignant glioma invasion in a three-dimensional model system. Primary glioblastoma (GBM) explants or multicellular spheroids were implanted into a three-dimensional collagen type I matrix. Individual tumor cells detached from the explants or spheroids and invaded the matrix, or invaded human fetal astrocyte spheroids. Treatment of implanted spheroids or explants with selective pharmacological inhibitors of ROCK was found to significantly decrease invasion. Inhibition of ROCK did not affect glioma cell or human astrocyte survival or proliferation. The identification of the Rho/ROCK pathway as being important for glioma cell invasion might be of therapeutic value in treating highly invasive glioblastoma.

The androgen receptor plays a critical role in both normal and neoplastic prostate growth. The first exon of the androgen receptor contains a polymorphic CAG repeat region that has been implicated in the development of prostate cancer. Epidemiologic studies suggest that shorter CAG repeats are associated with an increased risk of prostate cancer. Interestingly, the length of the CAG repeat region in the androgen receptor is inversely correlated with the transactivational competence of the receptor. This provides a biologically plausible mechanism by which the more active androgen receptors with shorter CAG repeats can contribute to prostate cancer. However, the effect of variation in CAG repeat length is unknown in the late-stages of the disease. / The primary goal of the work presented in this thesis was to determine the effects of different AR CAG repeat lengths in a late-stage prostate cancer environment where androgen receptor mutations are more common and may override the functional attributes of short CAG repeats. To that end, we have assessed the effects of CAG repeat lengths in a common prostate cancer mutant androgen receptor Thr877Ala. Our results suggest that the Thr877Ala mutant exhibits different transactivation trends than the wild-type receptor with respect to CAG repeat length. These data may indicate that, although CAG length may have a significant effect in wild-type receptors and in early disease, the activity of mutant receptors found in later stages of prostate cancer may limit the CAG effect.