Conventional two-dimensional glass and plastic in vitro cell culture systems potentially lack an accurate representation of the in vivo reality of three-dimensional cell locomotory processes; an important loss of valuable information or even misinformation in understanding and interpreting underlying cell behavioral mechanisms could result. This thesis uses a dynamic quantification approach of human tumor cell locomotory behavior in a three-dimensional collagen gel matrix in conjunction with time-lapse videomicroscopy and computer digitization. This approach better approximates physiological in vivo cell behavior by allowing cell trajectory analysis and expression of phenotypic characteristics previously not observed in two-dimensional culture systems or end point measurements. The main objective of this thesis is to determine whether there are quantifiable differences between benign, malignant, and normal cell locomotory behavior that can be measured in a significant and useful manner. The second objective of this thesis is to determine whether quantifying cell locomotory behavior can potentially reveal the existence of functional subpopulations within a heterogeneous tumor population. Further study of such a three-dimensional system can expand our insight into the metastatic and invasive processes of cancer and potentially contribute in assessing neoplasm aggressiveness and even patient morbidity.

Our study aimed at understanding the regulation of parathyroid hormone related peptide (PTHRP) by androgens in prostate cancer. Although PTHRP has been identified as a factor responsible for malignancy-associated hypercalcemia, it is known to be widely expressed in normal tissues where it regulates cellular growth and differentiation. PTHRP binds with equal affinity and activates the membrane-embedded G protein-coupled PTH/PTHRP receptor present on the surface of target cells such as bone and kidney. Upon receptor binding several signaling pathways are activated including the adenylyl cyclase and the phospholipase C pathways. / A number of growth factors and steroid hormones have previously been shown to regulate PTHRP production. To study the regulation of PTHRP by androgens, we used a late stage human prostate cancer cell line that is androgen non-responsive, PC-3 cells. In addition, we used an androgen responsive cell line, transfected with a functional androgen receptor, PC-3T cells. Results obtained by Northern blots and immunoradiometric assays demonstrated that androgens inhibit PTHRP expression in this human prostate cancer system. Male Balb/c nu/nu mice, injected subcutaneously with PC-3 cells, developed larger tumors than experimental animals inoculated with PC-3T cells. Moreover, castration of the host animals resulted in the substantial increased in PC-3T tumor volumes. Immunohistochemical studies indicated that PTHRP expression was inhibited in experimental PC-3T tumors and that castration of animals increased PTHRP production. In conclusion, our study demonstrated that androgens down regulate PTHRP production in prostate cancer cells in vitro and that prostate tumor cells producing less PTHRP exhibited a reduction in tumor volume in vivo.

Background: Young BRCA mutation carries with a history of breast cancer often inquire about the impact of pregnancy upon their risks of cancer recurrence and survival. Methods: We identified 128 BRCA carriers who were diagnosed with breast cancer while pregnant or who became pregnant after breast cancer diagnosis. Women were matched to 269 controls. Women were followed from the date of breast cancer diagnosis until the date of death. The Kaplan-Meier method and a left-truncated Cox proportional hazard model were used to estimate 15-year survival rates. Results: The adjusted hazard ratio associated with 15-year survival for women diagnosed with breast cancer who were or became pregnant after breast cancer diagnosis, compared to women who did not become pregnant was 0.76 (95% CI 0.31 to 1.91 p = 0.56). Conclusion: Pregnancy concurrent with or after a diagnosis of breast cancer does not appear to adversely affect survival among BRCA1/2 mutation carriers.

The insulin-like growth factor (IGF) pathway consists of two ligands (IGF-I and IGF-II), two receptors (IGF-IR and IGF-IIR) and six IGF binding proteins (IGFBP-I through -6). There is considerable evidence from both laboratory and population studies that IGF physiology is relevant to neoplastic growth. For example, it has been shown that IGF-I and/or IGF-II act as mitogens and anti-apoptotic agents for both normal and malignant cells by binding to the IGF-IR and activating downstream signalling pathways. Consistent with this data, IGF-IR inhibition by a variety of strategies inhibits cancer cell proliferation and/or induces apoptosis both in vitro and in animal models of neoplasia. Furthermore, epidemiological studies have demonstrated a positive correlation between serum IGF-I levels and risk of subsequent cancer. Classically, the IGFBPs were considered to be growth inhibitors, as they had a well-defined role in sequestering the mitogens IGF-I and IGF-II, therefore preventing binding and subsequent activation of mitogenic and anti-apoptotic pathways downstream of the IGF-IR. However, increasing evidence indicates that under certain conditions, IGFBPs can act as growth stimulators, and both IGF-dependent and -independent mechanisms have been proposed. / Although the roles of the IGFs, IGF-IR and IGFBPs in cancer have been studied extensively, this thesis describes several new links between IGF physiology and neoplasia. In the first section, we demonstrate that IGF-I can attenuate growth inhibition and apoptosis induced by a class of drugs called COX-2 inhibitors in BxPC-3 pancreatic cancer cells. This effect could be attributed to opposite influences of IGF-IR signalling and COX-2 inhibitors on activation of Akt, with IGF-IR signalling increasing activity and COX-2 inhibitors decreasing activity. In the second section, we demonstrate that in 184htert cells, an immortal but untransformed breast epithelial cell line, COX-2 inhibitors can induce IGFBP-3 expression. We go on to show that IGFBP-3 can inhibit growth of this cell line in an IGF-dependent manner, and speculate that this action of COX-2 inhibitors may be relevant to data linking use of this class of drugs to decreased breast cancer risk. In the third section, we demonstrate that the expression of IGFBP-2 in U251 glioma cells is inhibited by the induction of the tumor suppressor PTEN. Furthermore, IGFBP-2 does not effect the growth of this cell line, indicating that published associations between tumor IGFBP-2 expression and grade of glioma may be a result of IGFBP-2 acting as a marker for loss of function of PTEN. In the fourth and final section, we demonstrate that in MDA-MB-231 breast cancer cells, over-expression of IGFBP-2 can enhance growth, indicating that the effect of IGFBP-2 on growth of neoplastic cells is tissue specific. Furthermore, antisense strategies targeting IGFBP-2 mRNA (antisense oligonucleotides and siRNA) can inhibit growth of IGFBP-2-expressing breast cancer cells both in vitro and in vivo. / Taken together, these results extend the existing body of evidence demonstrating that IGF physiology contributes to neoplastic growth, and suggest that strategies to inhibit IGF-IR signalling and/or IGFBP-2 expression may have therapeutic value for some types of cancers.

In the past decade, new concepts have emerged on the role of adhesion molecules in the communication, motility, differentiation, and survival of cells in the body. The traditional view on the role of cell adhesion molecules in cell differentiation and tissue architecture has changed following the discovery that some adhesion proteins, including CD44, are involved in tumor progression in both animal and human tissues. CD44 is a glycoprotein which has differently spliced isoforms with various combinations of up to 9 variant exons numbered v2 to v10. The aim of this study was to characterize CD44 transcripts associated with colorectal neoplasia. The hypothesis is that discrete transcripts of CD44 are generated during the progression from normal to carcinomatous colonic epithelium, contributing to determinant changes in cell adhesion. Our results showed: (i) a high level of expression of CD44 v8-v10 transcripts in all tumor cells by in situ hybridization technique, (ii) 4 transcripts of CD44v7 with 650, 740, 1000, and 1150 bases in RNA extracted from tumor cases and studied by RT-PCR followed by Southern blotting, and (iii) the 1150 transcript was the only one found in both carcinoma and polyps. This transcript has therefore the potential to be used as an early marker of progression in colorectal neoplasia. It is reasonable to suggest that these new transcript species generate protein isoforms with different adhesion properties, as compared to standard CD44.

Cytogenetic studies to identify chromosomal aberrations in ovarian tumors allow for a better understanding of tumor biology. Fifteen ovarian tumors (1 benign, 7 borderline, and 7 malignant tumors) and one normal control were successfully characterized using conventional cytogenetic methods. Among simple karyotypic changes, trisomy 10, not previously reported in ovarian cancer, was confirmed with fluorescence in situ hybridization (FISH). This latter may be considered as a single causative event or as one among many other chromosomal alterations, being both associated with ovarian tumorigenesis and tumor progression. Among complex karyotypic abnormalities, one cell one (OV90) was further characterized using combined GTG banding and fluorescence in situ hybridization (FISH) techniques with painting probes from all the chromosomes, and chromosome specific centromeric probes, and a 10p telomeric probe derived from a half-YACs. The short arm of one chromosome 3 was deleted and replaced by a homogeneous staining region (HSR) which was demonstrated to be originated from amplification of yet unidentified genes on chromosome 22. We also identified a complex chromosome rearrangement (CCR) involving chromosome 9, 10 and 17. A dicentric chromosome, dic(9;17)(p11;p11), and a derivative chromosome, der(10)del(10)(pter)t(10;17)(p15;p11.1p11.2 or p13) were demonstrated, with a deletion in the short arm of chromosome 9, and a partial deletion of chromosome 17p. A translocation of chromosome 10p telomere to chromosome 1p was also observed. In addition, partial trisomy of chromosome 13q14-qter was also characterized and considered to result from a duplication and a pericentric inversion. These chromosomal aberrations were. identified in all cells analyzed and in each cell culture despite long-term cell culture, repeated freezing and thawing. These abnormalities are thus probably responsible for tumorigenesis, tumor evolution or in vitro survival of cancer cells. Further characteriz

Estrogen modulates growth and function of female reproductive tissues, such as uterus and mammary gland, by eliciting an array of biochemical responses and is linked to mammary carcinogenesis. To further understand the involvement of estrogens in complex physiological processes, we sought to increase the spectrum of genes regulated by estrogens. We cloned a novel 3.719 Kb cDNA from rat uterine tissue, termed steroid sensitive gene-1 (SSG1), encoding a nuclear protein of 386 amino acids and demonstrated that it is regulated by 17beta-estradiol in the uterus and mammary gland. In the rat uterus, SSG1 mRNA is down-regulated by 17beta-estradiol in time- and dose-dependent manners. In the mammary gland, chronic 17beta-estradiol treatment resulted in a significant accumulation of the SSG1 protein, which was dependent on the presence of elevated levels of 17beta-estradiol. SSG1 was consistently over-expressed in estrogen-dependent 7,12-dimethylben(a)anthracene-induced rat mammary tumors which may be a consequence of an altered hormonal environment. In rat and human mammary tissue, SSG1 protein was localized to the myoepithelial cells of the mammary ducts and to the smooth muscle cells of the vasculature. In human mammary tumors SSG1 was predominant in the epithelial compartment. In males, we demonstrate that SSG1 protein expression, immunolocalized to the prostatic smooth muscle cells and to the smooth muscle cells of the vasculature, is dependent on the presence of androgens. We conclude that SSG1 expression is regulated by steroid hormones in both female and male reproductive systems. We propose that SSG1 is a target for both estrogen and androgen modulation and that it may be implicated in estrogen functions related to mammary carcinogenesis.

CDP/Cux is a transcription factor involved in the control of cellular differentiation and proliferation. It contains four DNA binding domains, three Cut Repeats (CR) and a Cut homeodomain (HD). The full-length p200 protein makes an unstable interaction with DNA via CR1CR2 and is responsible for the CCAAT displacement activity of CDP/Cux. A more stable DNA binding activity could also be detected. This particular activity increased as cells progress from G1 to S phase of the cell cycle following two events. One event is the dephosphorylation of CR3HD by the by the cdc25A phosphatase. The other event involves the proteolytic processing of the full length CDP/Cux protein into a p110 isoform. At the transcriptional level, the p110 protein is able to repress the P21waf1/ Cip1 gene promoter and to activate the DNA polymerase alpha gene promoter as well as other S phase specific genes. In human uterine leiomyomas, an increase in the steady state levels of p110 was observed compared to the normal surrounding myometrium. / The goal of my project was to identify the protease involved in the proteolytic processing of CDP/Cux. I showed that the "lysosomal" cysteine protease cathepsin L translocates to the nucleus in a cell cycle dependent manner and was able to cleave p200 CDP/Cux into the p110 form. I also demonstrated that this protease was translated at downstream AUGs to produce truncated proteins devoid of signal peptide. These truncated cathepsin L species were unable to enter the endoplasmic reticulum and be routed to the lysosomes. In transformed cells, I found that CDP/Cux processing was increased and no longer regulated in a cell cycle dependent manner. This correlated with the enhanced expression and nuclear activity of cathepsin L. I also identified a novel shorter CDP/Cux isoform, which contains only CR3HD as DNA binding domains and is translated from an mRNA initiated within the intron 20 of CUTL1. Interestingly, the intron20-initiated mRNA was distributed in a tissue specific manner, and was expressed only weakly or not at all in normal breast tissue and in human mammary epithelial cells. However, it was detected in many breast tumor cell lines and invasive lobular carcinomas. These results indicated that in cancer, several mechanisms could generate shorter active CDP/Cux isoforms, and that these amino-terminally truncated CDP/Cux proteins play a role in tumorigenesis.

Retinoids are derivatives of vitamin A with demonstrated therapeutic potential for the treatment of breast cancer. The efficacy of retinoids in vitro and in vivo correlates with the expression of the estrogen receptor alpha (ERalpha). The role of ERalpha in mediating RA-induced sensitivity is not understood and is further complicated by the recent discovery of ERalpha. This dissertation explores the transcriptional, as well as proliferative, response to RA in human breast cancer cells expressing ERbeta or ERbeta. First, ER-negative breast cancer cells were stably transduced with ERalpha-deletion mutants using retroviral technology. We compared the effect of the ERalpha wild-type, ERalpha-deletion mutants or the parental ER-negative cells on transcriptional activity from the RARbeta2 promoter, a gene regulated by retinoids and potentially involved in retinoid-mediated growth inhibition. We observed that expression of ERalpha suppressed basal expression of the RA-responsive gene RARbeta2, while allowing it to be strongly induced by RA. Repression of basal RARbeta2 transcription was confirmed by transient expression of a reporter plasmid containing the RARbeta2 minimal promoter. We further determined that RARbeta2 induction required the N-terminal AF-1 containing region of ERalpha, including the DNA-binding domain, but was independent of the C-terminal ligand-binding domain. The effect of ERalpha was specific for RAR-mediated transcription and did not alter transcription from vitamin D or thyroid hormone response elements. Moreover, the cross-talk between ERalpha and RAR was not mediated by sequestration of a number of common co-regulators. To characterize the growth and transcription effect of ERbeta on retinoid-mediated pathways, we generated stable transfectants using this isoform. Significant RA-mediated growth inhibition was observed in the ERbeta-positive cells and not in the parental ER-negative cells. Furthermore, RA altered ERbeta-me

Adhesion molecules are present throughout embryonic development and during several physiological and pathological processes. Biliary glycoproteins (Bgp) are cell adhesion molecules that belong to the carcinoembryonic antigen (CEA) family, a member of the Ig supergene family. In an attempt to develop a relevant mouse model, we characterized the gene organization, regulation and expression patterns of the mouse Bgp1 gene. The mouse Bgp1 gene is down-regulated upon tumor transformation. This phenomenon has been correlated with a change in the methylation state of the promoter region of the mouse Bgp1 gene. Two other Cea-related genes, called the Bgp2 and Bgp3 genes, that also belong to the mouse Bgp gene family according to their deduced amino acid sequences and phylogenetic analyses, have also been characterized. Thus, three Bgp genes exist in the mouse genome while a unique BGP gene is found in the human genome. We have demonstrated that the Bgp2 gene product can also act in vitro as an alternative receptor for the mouse hepatitis virus. However, the expression pattern of the mouse Bgp2 gene is less abundant in the colon which is the natural route for MHV infection. Interestingly, the mouse Bgp3 gene presents a gene organization that resembles that of the pregancy specific (Psg) genes, another Cea-related gene subgroup. Expression patterns as well as phylogenetic studies suggest that the Bgp3 gene may represent an evolutionary link between the Cea subgroup and the Psg subgroup. The question remains open regarding the role of Bgp either during embryogenesis and during tumor development. Our studies on the different mouse Bgp gene expression and organization will render a gene ablation approach possible.