Gap junctional intercellular communication (GJIC) allows for the transport of small signaling molecules between adjacent cells. Gap junctions and their constituents, connexins, are often impaired in cancer. Restoration of connexins and GJIC in cancer cells leads to reversal of the transformed phenotype, reduction of the rate of cell growth, and inhibition of in vivo tumorigenicity. In addition, restoration of connexin43 (Cx43), one of the most abundant connexins that is often reduced in cancer, allows for the transfer of chemotherapeutic agents among neighboring cells, a phenomenon known as the "Bystander Effect". In chapter 2, I'm reporting evidence that induction of Cx43 and GJIC with cyclic-AMP (cAMP) can enhance the cytotoxic effect of the prodrug ganciclovir following infection of breast cancer cells with an adenovirus expressing the ganciclovir-activating enzyme viral thymidine kinase (vTK). This induction may provide a therapeutic advantage in suicide gene therapy due to the bystander effect when a small proportion of the cell population is expressing vTK. In chapter 3, I'm reporting that breast tumor tissue samples from patients had low to undetectable Cx43 levels, in contrast to their matched normal tissues. In addition, Cx43 protein and RNA levels were undetectable in a panel of human breast cancer lines and in rat breast tumors induced by the carcinogen dimethylbenz[a]anthracene. Together, these observations indicate that the loss of Cx43 is a common marker of cancer cells. In chapter 4, I examined the hypothesis that Cx43 downregulation occurs primarily at the transcriptional level. To determine the mechanisms behind the transcriptional regulation of Cx43, the human Cx43 promoter was investigated. Overexpression of the H-Ras oncogene in NIH3T3 cells leads to the induction of the human Cx43 promoter activity, as well as Cx43 RNA and protein levels. This stimulation involves the MEK-ERK pathway downstream of H-Ras. The promoter sequence responsible for

The Bcr-abl translocation arises from a reciprocal translocation between chromosomes 9 and 22 and results in the augmentation of the tyrosine kinase activity of c-Abl. Chronic myelogenous leukemia (CML) is one of several hematological malignancies associated with Bcr-abl expression. The pathogenesis of CML, associated with P210Bcr-abl, is bi-phasic consisting of an initial chronic phase followed by a severe terminal phase referred to as acute blast crisis. The chronic phase of the disease is characterized by granulocytic hyperplasia but in which normal hematologic maturation is still intact. Patients ultimately enter the terminal blast crisis where hematologic maturation is lost, resulting in the accumulation of immature blast cells and a severe immuno-compromised state. Progression to the blast terminal phase is associated with genomic instability demonstrated by the accumulation of genetic and cytogenetic abnormalities. Results from in vitro cell line systems expressing Bcr-abl have suggested that the loss of cell-cycle arrest and induction of apoptosis, as a result of genotoxic stress, might be responsible for this phenotype. In this study, I utilized a transgenic mouse model which expresses P190Bcr-abl to extend those observations to an in vivo model for leukemia. I observed normal cell-cycle arrest and induction of apoptosis following the induction of DNA damage. However, using the Big Blue in vivo mutagenesis mouse assay system, I evaluated genomic instability in P190Bcr-abl mice by measuring mutation frequencies in vivo. I observed an increase in mutation frequencies in spleens and kidneys from P190Bcr-abl mice. This Bcr-abl-induced mutator phenotype may explain the inherent genomic instability associated with the progression of CML and other diseases associated with the expression of activated tyrosine kinases.

Clinical cancer treatment has focused on the use of cytotoxic agents and/or radiation therapy that target both tumor and normal cells. Consequently, current cancer treatments with chemotherapeutic agents are subject to limitations associated with high toxicity and resistance. The cysteine proteinases cathepsin B and L have been linked to the invasive steps during the metastatic process and could therefore provide new targets for drug development. / The present work describes our results with a murine Lewis lung carcinoma model which consists of two cell lines, H-59 and M-27, with different patterns of metastasis in vivo. Using this model, we found that the cysteine proteinase inhibitor, E-64, significantly inhibited the invasive/metastatic properties of the liver colonising cell line, H-59 both in vitro and in vivo. Because IGF-1R was identified as a critical mediator of matrix metalloproteinase-2 (MMP-2) synthesis, invasion and metastasis in H-59 cells, the possibility that the cysteine proteinases interfered with receptor for type 1 insulin-like growth factor (IGF-1R) turnover thereby reducing invasion was subsequently investigated. / To elucidate more specifically the role of cysteine proteinases in the process of liver metastasis, cathepsin L expression was inhibited in the H-59 cells by transfection with a plasmid vector expressing a 300 by cathepsin L cDNA fragment in the antisense orientation. To further investigate the link between IGF-1R expression levels and invasion in these cells, MMP-2 production and activity were investigated. In cathepsin L antisense transfected H-59 cells reduced MMP-2 levels and activity as compared to controls were observed. Together our results suggest that the cysteine proteinases, cathepsin L in particular may regulate the metastatic potential through a role in IGF-1R turnover. The present results provide evidence that metastatic carcinomas which utilize cysteine proteinases for invasion could potentially be responsive to antimetastatic treatment with cysteine proteinase inhibitors. (Abstract shortened by UMI.)

The retinoblastoma tumour suppressor family plays an important role in the regulation of cellular growth in the cell cycle, growth arrest, and differentiation. The RB family includes pRB, p107, and p130 which interact primarily with two sets of transcription factor families, E2F and DP, resulting in the inactivation of E2F/DP transcriptional activity. The RB family members are targets for DNA tumour viral oncogene products such adenovirus E1A proteins which interact with all members, and displace E2F/DP heterodimers, resulting in the activation of E2F-dependent transcription. The binding of pRB was found to be absolutely required for EIA-mediated transformation, however, there is no correlation between E2F activation and transforming efficiency because some E1A mutants which failed to interact with pRB induced E2F at high levels presumably due to interaction with p107 and p130. These results suggest that pRB regulates E2F activity differently from p107 and p130. Analysis of mutants in conserved region 2 (CR2) of the E1A protein, including the (D)LXCXE conserved motif showed that the integrity of these conserved residues is important for interactions with both pRB and p130, but no single site mutation diminished binding of p107. Thus interactions with multiple members of the RB family appeared to be required for full activation of E2F and stable cell transformation by E1A proteins. / In undifferentiated cells such as C2C12 and P19 mouse cells, E2F is primarily complexed with p107/cyclinA/E/Cdk2. It was show that formation of pRB/E2F complexes is specifically induced during cell differentiation. In addition, p130/E2F complexes are also generated. We identified a novel slowly-migrating p130/E2F complex which is present not only in terminally differentiated cells, but also in growth-arrested cells. This complex is found in high amounts in Go/G1 phases, it disappears near the G1/S transition, and reappears in mitosis. Analysis of the content of these complexes showed that they contain cellular protein(s) which interact with p130 in a fashion similar to the CR2 region of E1A. Characterization of the novel E2F complex by antibody supershift assays revealed the presence of a known RB-binding protein, RBP1 which seems to be part of large family of cellular proteins interacting with p130 and which may be involved in the represssion of basal promoter activity of E2F regulated genes.

No description available.

Human prostate cancer is one of the most common malignancies affecting men. It is associated with a high degree of mortality and morbidity due to the development of non-skeletal and skeletal metastases. A common complication in patients suffering from prostate cancer is the development of hypercalcemia of malignancy. While determination of PSA and PSP-94 levels can serve as diagnostic/prognostic markers, PSP-94 can also serve as a therapeutic agent. The efficacy of PSP-94 to block tumor progression and hypercalcemia of malignancy was tested in our syngeneic in vivo rat model of prostate cancer. Rat prostate cancer Mat Ly Lu cells were transfected with full length cDNA encoding PTHrP [Mat Ly Lu-PTHrP] which is known to be the main factor responsible for hypercalcemia of malignancy. Mat Ly Lu-PTHrP cells were inoculated subcutaneously into the right flank or via intracardiac injection into the left ventricle of male Copenhagen rats. Animals were treated with different doses of PSP-94. Tumor volume, time of hind-limb paralysis, plasma calcium and plasma and tumoral PTHrP levels were determined. PSP-94 caused a significant delay in the development of hind-limb paralysis, reduction in tumor volume, plasma calcium levels, plasma and tumoral PTHrP levels as compared to control animals receiving vehicle alone. These effects were due to the induction of tumor cell apoptosis. In conclusion, our studies illustrate the ability of PSP-94 to block prostate cancer growth and skeletal metastases.

Estrogen (17-beta estradiol) and its receptor (ERalpha) have important physiological roles and are well implicated in human breast cancer, but less is known about the function of estrogen-related orphan nuclear receptor alpha (ERRalpha). The close kinship between ERalpha and ERRalpha and the existing, but yet to be fully characterized, interplay between ERalpha and erbB2 (Neu) protooncogene signaling pathways, suggest that ERRalpha may also play a significant role in breast cancer. Thus, the focus of the current study was to determine the extent of ERRalpha cross talk with ERalpha, its physiological role, as well as its possible implication in erbB2-driven mammary tumorigenesis. Using a well characterized erbB2 mouse model of human breast cancer where tumorigenesis occurs following a long latency period, we generated ERRalpha deficient mice (ERRalphaKO) expressing an activated erbB2 oncogene.

The purpose of this study was to determine among surgically treated non-metastatic breast cancer patients in the province of Quebec the distribution of the time between surgery and post-operative radiation therapy (RT) as well as secular trends and other factors influencing waiting time. Using administrative records, I identified between 1992 and 1998 29,105 episodes of breast cancer and 17,704 of these contained an indication of receiving RT Hierarchical linear regression models were used to identify predictors of waiting time. / The number of cases of breast cancer increased by 5.5% per year while the number of those receiving RT increased by 9%. Median post-surgery waiting time was 75 days in 1992 and by 1998 it had increased by 63% (95% Confidence Interval (CI) 35%--97%) among patients not requiring chemotherapy. In patients receiving chemotherapy, post-chemotherapy waiting time increased from 21 to 30 days (35% increase between 1998 and 1992 (95% CI -3%--88%)). In addition to a significant variability of waiting time according to radiation therapy centre, predictors of shorter waiting times were earlier year of treatment, localised cancer stage, breast conserving surgery, early consultation with a radiation oncologist, being operated in a centre with a radiation therapy facility, living close to a radiation therapy facility, and living in a higher socio-economic area. / In conclusion, waiting time to start of radiation therapy after localised breast cancer increased substantially in Quebec from 1992 to 1998. Possible explanations include increased demand, insufficient resources and changes in the indications for breast conserving surgery and RT.

Microarray expression analysis was applied as an approach for identifying cancer-related genes on chromosome-X (CHR-X) in epithelial ovarian cancer (EOC). The Hu6800 and U133A GeneChipsRTM were used to evaluate the expression of 446 CHR-X genes in an in vitro EOC model system comprising 4 EOC cell lines and 12 primary cultures of normal ovary surface epithelia. Fifty-one new candidate CHR-X genes were identified in addition to 49 genes previously implicated in cancer. Many genes map to regions with frequent genetic aberrations in EOC tumours, or interact with the known EOC tumour suppressors BRCA1 and BRCA2. Candidate genes described in this study may provide novel markers for histopathological subtypes, or the tumourigenic potential of EOC tumours. The X-inactive-specific-transcript (XIST) was absent in two highly tumourigenic EOC cell lines, TOV21G and TOV112D. XIST mRNA is important for the stability of X-chromosome-inactivation (XCI), as its absence destabilizes the silencing of genes on the inactive-X. Aberrant bi-allelic expression of FHL1, a gene subjected to XCI was detected in the cell line TOV21G but not in the cell line TOV112D. Genotyping assays using polymorphic microsattelite markers suggested that TOV21G has retained heterozygosity of CHR-X. The majority of alleles tested for TOV112D were consistent with loss of heterozygosity of CHR-X. Taken together these findings are consistent with two proposed mechanisms mediating XIST loss-of-expression in cancer: (1) Duplication of the active-X followed by loss of the inactive-X (TOV112D); or (2) Reactivation of the previously inactive-X (TOV21G).

<p> Current research into the detection and characterization of circulating tumor cells (CTCs) in the bloodstream can be used to assess the threat to a potential cancer victim. We have determined specific goals to further the understanding of these cells. 1) Full automation of an algorithm to overcome the current methods challenges of being labor-intensive and time-consuming, 2) Detection of single CTC cells amongst several million white blood cells given digital imagery of a panel of blood, and 3) Objective classification of white blood cells, CTCs, and potential sub-types. </p><p> We demonstrate in this paper the developed theory, code and implementation necessary for addressing these goals using mathematics and computer vision techniques. These include: 1) Formation of a completely data-driven methodology, and 2) Use of Bag of Features computer vision technique coupled with custom-built pixel-centric feature descriptors, 3) Use of clustering techniques such as <i> K</i>-means and Hierarchical clustering as a robust classification method to glean insights into cell characteristics. </p><p> To objectively determine the adequacy of our approach, we test our algorithm against three benchmarks: sensitivity/specificity in classification, nontrivial event detection, and rotational invariance. The algorithm performed well with the first two, and we provide possible modifications to improve performance on the third. The results of the sensitivity and specificity benchmark are important. The unfiltered data we used to test our algorithm were images of blood panels containing 44,914 WBCs and 39 CTCs. The algorithm classified 67.5 percent of CTCs into an outlier cluster containing only 300 cells. A simple modification brought the classification rate up to 80 percent of total CTCs. This modification brings the cluster count to only 400 cells. This is a significant reduction in cells a pathologist would sort through as it is only .9 percent of the total data.</p>