Artificial cells microencapsulation have a number of potential areas of application. Studies showed that lowering of tyrosine level could inhibit the growth of melanoma. However, at present there is no practical method to lower the tyrosine level in humans. We have therefore devised novel methods as follows. (1) Microencapsulation of tyrosinase for the removal of systemic tyrosine by oral administration. Characterization, optimization, and feasibility studies were carried out to test the therapeutic potentials. In temperature and pH studies, the encapsulated tyrosinase maintained higher enzyme activity than the free enzyme in solution. The in vivo studies showed that daily oral administration of encapsulated tyrosinase by itself for about 3--5 days could lower the body tyrosine level. (2) A novel polyhemoglobin-tyrosinase preparation for intravenous injection can rapidly lower the body tyrosine level after one intravenous injection. In this form, the enzyme is covered by hemoglobin molecules and therefore has less immunological properties. Furthermore, polyhemoglobin is an oxygen carrier and being in a solution, it can more readily reach the narrower capillaries of the melanoma cancer cells than red blood cells and can therefore bring more oxygen for radiation therapy. Our in vitro studies showed that this novel polyhemoglobin-tyrosinase preparation inhibited the growth of melanoma cells in culture. (3) A combination of two intravenous injections of polyhemoglobin-tyrosinase with 3 times a day oral administration of encapsulated tyrosinase could immediately lower the body tyrosine and maintained this low level as long as the oral administration was continued.

Metastasis initiation is a complex process encompassing numerous steps. To identify molecular alterations associated with early and late stages of metastasis, we used high throughput screening techniques. Early events in metastasis were investigated by differential proteomic analysis of lymph node-negative and positive breast cancer samples. Two candidate biomarkers (DCN and HSP90B1) were discovered and further validated through tissue microarray analyses. To examine late events in metastasis formation, we prospectively evaluated genomic differences between disseminated tumour cells in bone marrow, and metastatic tumour cells obtained from computed tomography guided biopsies of bone metastases. Results indicate that specific subsets of genes are required for breast cancer cells to initiate bone metastases. Discovery of proteomic and genomic alterations specifically associated with metastases may yield biomarkers capable of stratifying patients into different risk categories. Proteins and genes identified in this work may form the foundation of a biomarker panel for metastatic risk assessment.

Metastasis initiation is a complex process encompassing numerous steps. To identify molecular alterations associated with early and late stages of metastasis, we used high throughput screening techniques. Early events in metastasis were investigated by differential proteomic analysis of lymph node-negative and positive breast cancer samples. Two candidate biomarkers (DCN and HSP90B1) were discovered and further validated through tissue microarray analyses. To examine late events in metastasis formation, we prospectively evaluated genomic differences between disseminated tumour cells in bone marrow, and metastatic tumour cells obtained from computed tomography guided biopsies of bone metastases. Results indicate that specific subsets of genes are required for breast cancer cells to initiate bone metastases. Discovery of proteomic and genomic alterations specifically associated with metastases may yield biomarkers capable of stratifying patients into different risk categories. Proteins and genes identified in this work may form the foundation of a biomarker panel for metastatic risk assessment.

Cellular adhesion interactions involved in invasion and migration are crucial to cancer metastasis. Neoplastic tissue explants were cultivated in three-dimensional collagen lattices and monitored for the detachment of coordinated locomoting tumor cell clusters. Blocking monoclonal antibody treatment (10ug/mL) against the $ beta sb1$ and $ alpha sb2 beta sb1$ integrin receptors on cluster surfaces allowed the functional evaluation of these receptors during metastatic invasion. Clusters in treated wells exhibited a loss in migratory persistence, reduction in cluster motility and a greater amount of "stops" during migration as compared to control wells for both anti-$ beta sb1$ and anti-$ alpha sb2 beta sb1$ respectively. These results suggest that $ beta sb1$ integrins, and more specifically, $ alpha sb2 beta sb1$ play a significant role in maintaining normal cell-cell and cell-matrix interactions during metastatic invasion. Preliminary investigations using the anti-cancer drug, Taxol, against clusters within collagen matrices revealed a significant decrease in the total distance traveled, average speed and persistence, implicating its efficiency against certain cancers. The use of 3-D collagen systems for cell communication studies on locomoting tumor cell clusters may reveal novel and potentially important mechanisms involved in metastatic invasion as well as facilitate future assessment of potential anti-neoplastic drugs.

Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the metabolism of folate and methionine. MTHFR catalyzes the conversion of 5,10 methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF). 5-MethylTHF is a co-substrate for homocysteine remethylation to methionine catalyzed by the vitamin B12-dependent enzyme methionine synthase. A common MTHFR variant, 677C → T substitution resulting in the conversion of an alanine to a valine residue, has been shown to be a thermolabile form of MTHFR and to have reduced activity (Frosst et al., 1995). Since many diverse cancer cells have been documented to be methionine-dependent in culture, the effects of MTHFR downregulation on cell survival of transformed cells was examined. In addition, the influence of the MTHFR 677C → T polymorphism on the sensitivity of transformed lines to antifolate drug treatment was studied. / To test the effect of decreased MTHFR expression on cell viability, we transfected antisense oligonucleotides (ASOs) complementary to the MTHFR mRNA into the colon carcinoma cell line SW620. (Abstract shortened by UMI.)

DNA tumor virus oncoproteins have evolved to regulate the p53 tumor suppressor. They must overcome p53-dependent cell cycle arrest and apoptosis, which would interfere with viral replication. Upon cellular stress, signalling to p53 takes the form of a large increase in the stability of the protein, by inhibition of the MDM2 protein which normally targets p53 for rapid degradation. Expression of the adenovirus type 5 early region 1 A (E1A) polypeptides can stimulate a quiescent cell to reenter the cell cycle and induces the accumulation of p53 protein and p53-dependent cell death under certain conditions. We mapped the regions of E1A necessary to induce p53 stabilization. Binding of either the p300 or pRb family of proteins can signal to p53, and these are the same activities required for E1A to push the cell to enter S phase. To replicate, the virus must combat this accumulation of p53, and we discovered that two other early viral proteins, E1B55K and E4orf6, can target p53 for ubiquitin-mediated degradation by the 26S proteasome. We found that when cells are infected with adenovirus, no significant accumulation of p53 occurs, and this is due to a large reduction in the half-life of the protein. E4orf6 and E1B55K each bind p53 and also bind each other, and we generated a series of E4orf6 mutations to study the regions necessary for this interaction. We identified a region at the amino terminus of E4orf6 that is the minimal domain permitting interaction with E1B55K. We also determined that binding E1B55K along with functions requiring most of the E4orf6 carboxy terminus have to be intact for E4orf6 to mediate p53 degradation. We tested the activity of the viral proteins in MDM2-deleted cells, and found that E4orf6/E1B55K don't require MDM2 to induce p53 turnover. A series of cellular proteins that interact with E4orf6 were discovered in our lab, and we identified this complex as Cullin 5, Elongin B, Elongin C, Rbx1, E4orf6 and E1B55K. This complex is very similar

Human carcinoembryonic antigen (CEA) is the prototypic member of a large family of highly related cell surface glycoproteins that includes CEACAM6 (formerly NCA) and CEACAM1 (formerly BGP). The extracellular domains of CEA/CEACAM6 are bound to the external surface of the plasma membrane through a glycosylphosphatidyl inositol (GPI) anchor and are over-expressed in more than 50% of all human cancers. In contrast, CEACAM1 contains extracellular, transmembrane and cytoplasmic domains, and its level of expression is down-regulated in human tumors of the colon and prostate. When over-expressed on the surface of various cell types in model systems, CEA/CEACAM6, but not CEACAM1, function as pan-inhibitors of cell differentiation and cell polarization and cause a distortion of tissue architecture. Anoikis is a quality control mechanism that must be inhibited in cancer cells for such a distortion to persist. This thesis presents data demonstrating that CEA/CEACAM6 over-expression on the surface of a variety of cell lines inhibited anoikis. The molecular basis for the inhibitory effects of CEA/CEACAM6 on both anoikis and differentiation is shown to be correlated with perturbation of the function of certain integrins. In contrast to CEA/CEACAM6, the expression of the CEACAM1 glycoprotein neither perturbed integrin function nor prevented anoikis and, consistent with this, inhibited tumor growth. As a conclusion, we propose that CEA/CEACAM6, but not CEACAM1, over-expression on the surface of cancer cells inhibits cell differentiation and anoikis through perturbation of integrin functions. These inhibitory effects could instrumentally contribute to tumor formation and progression.

Urinary plasminogen activator (uPA), a member of the serine protease family, is implicated in the progression of various cancers including breast cancer due to its ability to provoke malignant cell invasion. uPA production is reported to be much higher in late stage, estrogen receptor (ER) negative breast cancer patients than those with benign hyperplasia. Since all existing evidence points to a role for uPA in breast cancer progression, exploring the mechanisms regulating its gene expression will be of immense value. Two human breast cancer cell lines were selected for this study. MDA-MB-231 represents late stage breast cancer. This cell line has high uPA expression and is highly invasive. In contrast, the MCF-7 cell line represents early stage breast cancer and fails to express detectable levels of uPA mRNA. I have demonstrated by methylation specific PCR (MSP) that the differential expression of the uPA gene in MDA MB-231 and MCF-7 cells closely correlates with the amount of methylated cytosines present within the uPA promoter in these cells. The observation that the DNA methylation status of the uPA promoter directly affects the expression of the uPA gene was then confirmed using an in vitro luciferase reporter assay. Results suggest that the accessibility of the transcription factor Ets-1 is limited by DNA methylation. I further reported increased demethylase (DMase) activity with decreased maintenance activity of methyltransferases (DNMTs), which together favor the generation of a hypomethylated uPA promoter in these highly invasive MDA-MB-231 breast cancer cells. Given the pivotal role of uPA in breast cancer progression, I then disrupted the function of uPA and studied its effects on cancer progression. The effects of an 8-mer synthetic peptide (A6) derived from the non-receptor-binding region of uPA were investigated. This peptide inhibits cancer cell invasion of both human (MDA-MB-231) and rat breast cancer cells (Mat B-III) using an in vitro cell invas

OV90, a spontaneously immortalized epithelial ovarian cancer (EOC) cell line, has been shown to contain a homogeneously staining region (HSR) originating from chromosome 22. To identify the amplified genes, differential gene expression was assessed using high-density oligonucleotide array (chip) technology. Genomic differential PCR and dot blot analysis showed that overexpression is consistent with gene amplification, as all sequences tested within the 1 Mb region of 22q11.21 are present in increased copy number. However, overexpression was not common, as only two of 69 EOC samples evaluated using the Hu6800 chip showed overexpression of a gene amplified in OV90, and three other EOC cell lines did not have increased expression in this region. This study tested the approach of chip technology to identify genes involved in gene amplification, in a rapid and reliable manner.

Over the past two decades, novel targets for anticancer agents have been identified. One such target, the epidermal growth factor receptor (EGFR) that is overexpressed in a large number of carcinomas including breast, ovarian, and prostate, is a marker for tumour invasiveness and poor prognosis. Agents of the quinazoline class have been developed that block EGFR-mediated signaling and induce antitumour activity in the clinic. However, the major deficiency of these compounds is that they are cytostatic agents that induce reversible antiproliferative activities. To circumvent these problems, we designed a novel tumour targeting approach termed "the combi-targeting" concept. This theory is based on the fundamental premise that compounds capable of interfering with multiple targets in the cancer cells are more efficient antitumour agents than the single-targeted ones. Thus, the "combi-targeting" concept proposes the design of molecules termed "combi-molecules" or TZ-I to not only bind to the tumour target but also to be allowed to degrade to another inhibitor I of the same target + a DNA damaging species TZ, leading to small molecules capable of repetitively blocking one target while damaging another. Using EGFR as a tumour target, the first TZ-I prototype SMA41, an anilinoquinazoline containing a triazene tail at the 6-position, was synthesized. We demonstrate herein that the compound enters the cell by passive diffusion, degrades under physiological conditions to yield an intact TK inhibitor ("SMA52") (I) + a short-lived DNA-damaging methyldiazonium species (TZ). In the EGFR-expressing human tumour cells, SMA41 (TZ-I) behaved as a binary targeted drugs with ability to: (a) inhibit EGF-induced receptor autophosphorylation, cell cycle progression and growth, (b) induce dose-dependent DNA damage, and (c) inhibit cell proliferation with greater potency than the released reversible inhibitor (I) both in vitro and in vivo. This work represents the