Modern cancer therapy has explored the use of enzymatically acting toxins coupled to specific binding proteins in an attempt to destroy specific cell populations. In light of these new therapies we have synthesized two hormonotoxins (HTs): (i) composed of the ribosome inactivating plant protein gelonin conjugated to luteinizing hormone (LH), (ii) gelonin conjugated to human chlorionic gonadotropin (hCG), both through a disulfide bond. / Characterization of both conjugates was carried out by SDS-PAGE analysis, radioimmunoassay and Western blotting (using polyclonal antibodies against both the hormone and the toxin). In addition, bioactivity of the hormonotoxins was determined by their ability to bind LH receptors in testicular membrane preparations. MA-10 cells showed a significant reduction in protein synthesis following a 24 hour exposure to the cytotoxic conjugates. / In vivo studies were carried out using the LH-gelonin hormonotoxin. Intravenous injections of $ sp{125}$I-HT into 28-30 day-old pseudopregnant mice and determination of radioactivity in the ovaries, kidneys, liver, thyroid, brain, and blood helped to establish preliminary results on the uptake of hormonotoxin by these tissues. Study of the cytotoxic effects of the conjugate in vivo in normal male mice (following a three week period of injection), revealed a significant reduction in testosterone production in animals which received the highest concentrations of hormonotoxin. In addition, treatment of animals with the separate components of the conjugate (same pattern and duration of treatment), established specificity of the effect exerted by the hormonotoxin. Treatment with the LH-gelonin hormonotoxin caused production of antibodies to both components of the conjugate. Results obtained from these studies have helped to establish the groundwork for the effects of HT in vivo in normal mice. Overall, the research on these HTs had provided encouraging information for their potential use as therapeutic agents. (Abstract shortened by UMI.)

B cell activation to immunoglobulin production requires both cytokine and contact-dependent help from activated CD4$ sp+$ T cells. Signals from multiple stimuli and thus integrated to produce an appropriate response. Intercellular adhesion molecule 1 (ICAM-1) has been implicated in the induction of cytokine responsiveness in small resting B cells. We now investigate the biochemical mechanism of ICAM-1 signaling in a B cell lymphoma, A20. Our results show that ICAM-1 ligation in these cells leads to growth arrest and upregulation of class II MHC. Crosslinking ICAM-1 results in activation of the src-related kinase p53/p56$ sp{ rm lyn}$, as well as Raf-1 and MAP kinases. Immunoprecipitation of ICAM-1 from unstimulated cells revealed that several tyrosine phosphorylated proteins are physically associated with ICAM-1. It is hoped that these data will lead to an understanding of the modulatory role of ICAM-1 in the immune response.

Several studies have shown that the retinoic acid (RA)-activated signaling pathway can interact with both the PKA and the PKC signaling pathways. The aim of this project was to investigate the mechanism of such an interaction in the differentiation of P19 and RAC65 murine embryonal carcinoma cells. Two activators and two inhibitors of both PKA and PKC were used in differentiation experiments. Of these eight modulators, only the PKA activator dibutyryl cyclic adenosine monophosphate (dbcAMP) and the PKC inhibitor sphingosine were found to significantly potentiate the effect of RA in the neuronal cell differentiation of these cells. However, neither of these two modulators showed a significant modulatory effect on RA-mediated transactivation. Moreover, neither dbcAMP nor sphingosine modulated the RA-induced expression of a retinoid-responsive gene (RAR$ beta$). In conclusion, the signaling pathways of PKA and PKC appear to modulate RA-induced P19 and RAC65 neuronal differentiation. We postulate that this interaction involves the products of genes expressed in response to these signaling pathways.

Ahi-1 was initially identified as a common helper provirus integration site on mouse chromosome 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to Myb proto-oncogene. By using long-range restriction mapping, we have mapped the Ahi-1 locus approximately 35 kbp downstream of the Myb gene. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region may activate a novel gene, apparently involved in tumor formation. In addition, another provirus integration site which was identified in 14% of immature T-cell thymomas induced by Mo-MuLV infection of MMTV${{ rm D} over C}$-Myc transgenic mice has recently been mapped within the Ahi-1 region. We have used exon amplification to identify genes that may cooperate with v-Ab1 or c-Myc in B- and T-cell transformation. We have indeed identified a novel Ahi-1 gene which encodes two major RNA species of 2 kbp and 5 kbp. The Ahi-1 gene is expressed in several organs of the mouse and rat and is highly expressed in brain and testis. One cluster of the proviruses was found to integrate 3$ sp prime$ of noncoding region of the Ahi-1 in an inverse transcriptional orientation. The Ahi-1 is highly conserved in evolution and encodes a 297 amino acid protein. The predicted Ahi-1 protein contains a SH3 domain and four potential SH3-binding sites, which function to mediate specific protein-protein interactions during signaling. The human homologue of the Ahi-1 gene has been cloned and mapped to chromosome 6q 23-24, and located approximately 330 kbp downstream of the Myb gene. The Ahi-1 is a novel gene which may play important roles in the signal transduction and tumor development. / Mis-2 and Mis-4 loci were identified as two new common provirus integration sites in Moloney MuLV-induced thymomas. Mis-2 has been mapped on mouse chromosome 10 and located 160 and 40 kbp downstream of Myb and Ahi-1 genes. The Mis-4 locus has been located on chromosome 15 at about 60 and 30 kbp downstream of Myc and Mlvi-4 genes. Myc RNA levels are elevated in tumors harboring a Mis-4 rearrangement compared with some of those with no Mis-4 rearrangement, indicating that provirus integration affected Myc expression by long-distance activation. Both loci may contain novel genes involved in tumor formation.

Cancers of the upper aero-digestive tract (UADT) rank as the fifth most common neoplastic disease worldwide. Two identified risk contributors are consumption of tobacco and alcohol. Among all other potential etiological factors, diet has long been recognized to play an important role in the development of cancers of the UADT. Data from a multi-centre, hospital-based case-control study conducted in Brazil were used to assess the association of dietary intake with the risk of cancers of the UADT. Dietary assessment was made in terms of estimated intake of nutrients, specific foods and food groups. After adjusting for the effects of alcohol and tobacco consumption as well as empirical confounders, protective effects against cancer of the mouth (Odds Ratio (OR) = 0.61, 95% confidence interval (95% CI): 0.4--1.0) and the pharynx (OR = 0.51, 95%CI: 0.3--0.9) were found for consumption of citric fruits. (Abstract shortened by UMI.)

Insulin-like growth factors have well characterized mitogenic and antiapoptotic effects in a number of normal and transformed cell types that are mediated through binding to the IGF-I receptor. IGF activity is modulated by at least six insulin-like growth factor binding proteins (IGFBPs) that bind IGFs with high affinity. Recently, it has been proposed that IGFBPs may play an important role in regulating apoptosis. This thesis provides evidence that IGFBPs induce apoptosis in vitro, and are associated with involution of ventral prostate and with regression of androgen-dependent and estrogen-dependent tumors in vivo. We show here that IGFBP-3 induces apoptosis in MCF7 breast cancer cells, and that apoptosis induced by the antiestrogen ICI 182,780 is mediated in part by increased IGFBP-3 accumulation. In the normal rat prostate, increased expression of genes encoding IGFBPs 2,3,4 and 5 is observed during apoptosis induced by castration, the antiandrogen bicalutamide, or the vitamin D3 analogue EB1089. A dramatic increase in IGFBP-5 gene expression is observed during castration-induced apoptosis in androgen-dependent Shionogi tumors. Inhibition of apoptosis in Shionogi tumors with calcium channel blockers attenuates castration-induced IGFBP-5 expression. These results indicate that IGFBP-5 gene expression is related to apoptosis, rather than being strictly under hormonal regulation. Increased expression of IGFBPs 2,3,4 and 5 is observed in regression of DMBA-induced rat mammary tumours induced by estrogen-ablation. Rapid induction of IGFBPs can limit access of IGFs to the IGF-I receptor and may therefore initiate induction of apoptosis. Taken together, these results demonstrate that IGFBPs play a role in apoptosis and suggest that strategies which target IGF pathways may augment the actions of hormone-targeting therapies for breast and prostate cancer prevention and treatment by enhancing apoptosis in tumors.

DNA methylation is a vital component of several mammalian genome functions such as X-inactivation, allele and tissue specific gene expression, parental imprinting, and condensation of transcriptionally inactive chromatin. Aberrant post developmental fluctuations in the levels of DNA methylation and patterns of DNA methylation are commonly observed in transformed or cancerous cells. Proper control over the process of DNA methylation appears to be essential for proper cellular function, however the regulation of the expression of DNA MeTase has remained only loosely elucidated and is still debatable. Chapter 1 presents the complete genomic structure of the human dnmt1 gene which provides an essential template to resolve the existing contradictions. Using this structure chapter 2 demonstrates that there are several different transcriptional control elements that influence the production of different dnmt1 transcripts from several different points of initiation. Furthermore it demonstrates that each of the transcriptional control elements can respond individually to oncogenic and tumor suppressor pathways. Proper control over dnmt1 expression in non transformed cells is achieved in a posttranscriptional manner. Chapter 3 of this thesis shows that the mechanism of the posttranscriptional control over dnmt1 expression is mediated by a conserved stretch of sequence contained within the 3' untranslated region of its mRNA. Evidence is presented that a labile protein factor existing in the G0-G1 part of the cell cycle, which is rapidly cleared in S-phase, mediates the degradation of the dnmt1 mRNA. The pharmacological potential of specific inhibition of dnmt1 expression in a whole organism tumor model is evaluated in Chapter 4. Inhibition of dnmt1 expression, by systemic administration of a phosphorothioate modified antisense deoxyoligonucleotide directed against dnmt1 mRNA, inhibits tumorigenesis with no overt toxicity. / The energetics of DNA demethylation suggested that it is an improbable event to occur in a cellular context, so alternative activities which repair DNA were rationalized to be the activities responsible for the observed demethylation events. Chapters 5 and 6 of this thesis reveal a true or bona fide DNA demethylase (dMTase) activity which catalyzes the reverse reaction of DNA MeTase. Chapter 5 describes a cDNA which encodes bona fide DNA dMTase activity and chapter 6 purifies a DNA dMTase activity, characterizes the reaction and identifies the products of the reaction as methanol and nonmethylated cytosine. The DNA dMTase cDNA cloned in chapter 5 is shown to be over expressed in cancer cells and that DNA dMTase activity can only be isolated from tumor tissue. Inhibition of DNA dMTase inhibits the tumorigenicity of cells on semi-solid media, indicating that over expression of dMTase is also a critical component of tumorigenesis. / Taken together the results presented in this thesis demonstrate that the DNA methylation machinery is a target for the development of anticancer therapeutics. The data presented here provide the rationale that direct inhibition of DNA dMTase or inhibition at molecular points of regulation are targets to be potentially exploited for therapeutic use.

Breast cancer is the second most frequent cancer in middle-aged women. Control of this disease requires identification of the various factors involved in the invasive-metastatic cascade, facilitating the subsequent development of relevant blocking compounds. / Tumor cell invasion is a complex sequence of events where cell adhesion molecules exert determinant roles. CD44 is a family of cell adhesion glycoproteins generated by alternative splicing of up to ten variant exons. Discrete CD44 isoforms are overexpressed in different human cancers, including breast cancer. CD44 is expressed on the plasma membrane of cells and binds mainly to hyaluronan. Hyaluronan is a negatively charged high-molecular-weight glycosaminoglycan conspicuously present in the extracellular matrix ant its concentration is increased at sites of tissue remodeling. / This study tested the hypothesis that CD44 and its main ligand, hyaluronan participate in the invasive properties of breast cancer cells. The hypothesis is based on the following rationale: the previously documented upregulation of CD44 expression in human breast cancer tumors, the role of CD44 in the invasive properties of nonepithelial cells in vitro, and the high concentrations of hyaluronan present in human breast cancer tumors. / The experiments were performed with different human breast cancer cell lines. In one cell line, CD44 and its v6 variant isoform participate directly in tumor cell invasion in vitro. Moreover, hyaluronan alters cell behavior in vitro, mainly by changing CD44 expression, adhesion and motility of breast cancer cells with high CD44 expression. In addition, it is shown that intratumoral injection of hyaluronan reduces the volume of tumors produced by orthotopically xenotransplanted breast cancer cells. / The contribution of these studies to the advance of knowledge in breast cancer is based on a role of CD44 and hyaluronan in the interactions between tumor cells and the extracellular matrix, offering an opportunity to develop a new therapeutic strategy.

The incidence of melanoma worldwide has been increasing steadily in recent years with an associated rise in mortality rates. If detected early, melanoma lesions can be surgically removed with wide excisional margins resulting in high cure rates. However, as the melanoma invades the deep layers of the skin at the primary site and subsequently metastasizes to the regional and distant nodes, the prognosis for the patient becomes progressively worse. As the melanocytic lesion progresses from a benign nevus to a highly malignant metastatic melanoma, it undergoes changes in protein expression which may be relevant to melanoma development. Among the proteins found to be upregulated during melanoma progression are the vitronectin adhesion receptor $ alpha sb{ rm v} beta sb3$ and the receptor for the proteolytic enzyme urokinase plasminogen activator (uPAR). The present work describes our results with two new animal models of human melanoma which were developed to study the cellular and molecular mechanisms involved in lymphatic metastasis of this disease. Each model consisted of two cell lines with different metastatic abilities. Using these models, a correlation was found between the adhesion of the melanoma cells to cryostat sections of human lymph nodes and their metastatic ability in nude mice. When compared to the parent cells, the lymph-node metastasizing melanoma cells were found to express increased levels of the cell surface vitronectin receptor (VNR) $ alpha sb{ rm v} beta sb3$ which was utilized to mediate the increased adhesion to lymph node. In addition to the VNR, the metastatic cells also expressed increased levels of the uPAR. To study the relationship between expression of these two receptors, $ alpha sb{ rm v}$ expression was suppressed with $ alpha sb{ rm v}$ antisense phosphorothioate oligonucleotides. In the antisense-treated cells a 9-fold reduction in $ alpha sb{ rm v}$ mRNA was seen with a corresponding 2.8-fold decrease in adhesion to vitronectin / In a parallel study, a model of human breast carcinoma was used to study the role of cell adhesion in breast carcinoma metastasis. A correlation was observed between cell adhesion to frozen sections of human lymph modes and metastasis in vivo. However, unlike the melanoma cells, the metastatic breast carcinoma cells were found to utilize receptors $ alpha sb3 beta sb1$ and $ alpha sb5 beta sb1$ to adhere to lymph node fibronectin suggesting that different adhesion receptors may be involved in lymph node metastasis of different malignancies.

This thesis explores the role of an adhesion protein, CD44, in the biology of colon cancer. CD44 glycoprotein is a main extracellular receptor for hyaluronic acid. A single CD44 gene composed of 20 exons encodes a variety of isoforms due to alternative splicing of 10 middle exons. Overexpression of CD44 and appearance of abnormal isoforms containing products of variant exons have been implicated in the origin and progression of cancer, including human colon carcinoma. / Of two main parts comprising the dissertation, the first one ("CD44 and the adhesion of neoplastic cells" by Z. Rudzki and S. Jothy) reviews the current state of knowledge on CD44, emphasizing its role in neoplastic processes. The second part ("Changes in CD44 expression during carcinogenesis of the mouse colon" by Z. Rudzki, L. LeDuy and S. Jothy) reports an experimental study of CD44 expression in a murine model of colon cancer. The tumors were induced by subcutaneous injections of a colon--specific carcinogen (1,2-dimethylhydrazine). CD44 expression was studied by RT-PCR/Southern blot and immunohistochemistry. The CD44 transcripts were generally strongly overexpressed in tumors compared with normal colon. Both neoplastic and normal colon samples exhibited the same complex array of transcript bands representing the standard molecule (CD44s) and its variant isoforms. Immunohistochemistry revealed marked heterogeneity of tumor staining, contrasting with a rather uniform mRNA overexpression. There was a significant tendency towards the progressive loss of CD44 immunoreactivity in larger and invading tumors. It is concluded that CD44 isoforms are globally overexpressed at an early, premacroscopic stage of colonic carcinogenesis. Expression of CD44 in the murine model of colon cancer shows some similarities to its human counterpart.