Acute promyelocytic leukemia (APL) is characterized by reciprocal chromosomal translocations invariably involving the retinoic acid receptor alpha (RARalpha) gene on chromosome 17. In the vast majority of APL cases, the RARalpha gene is fused to the PML gene on chromosome 15, generating the fusion PML-RARalpha. The chimeric PML-RARalpha protein plays a critical role in APL leukemogenesis. Retinoic acid (RA) can overcome the differentiation block conferred by PML-RARalpha and induce APL cells to terminally maturate into granulocytes. However, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones derived from the human APL cell line, NB4. These resistant subclones display altered RA-binding and reduced transactivation of retinoid response elements (RAREs) upon RA treatment. In the subclone R4, we identified a point mutation changing a leucine to praline in the ligand binding domain of the fusion PML-RARalpha protein that causes a loss of ligand binding. The mutant PML-RARalpha protein retains its ability to heterodimerize with RXRalpha and bind to RAREs. Thus, it functions as a dominant negative transcriptional inhibitor of the coexpressed wild-type RARalpha. The mutant PML-RARalpha has impaired RA-dependent interaction with receptor coregulatory factors, SMRT and ACTR. Histone deacetylase inhibitor, Trichostatin A (TSA), in combination with RA can overcome the dominant negative activity of the mutant PML-RARalpha in R4 and relieve transcriptional inhibition on RAREs. Further, TSA potentiates and partially restores RA-induced differentiation of NB4 and R4 cells respectively. When we examined the interaction of specific cofactors with retinoid receptors in NB4 cells compared to that in RA-resistant cell lines, we isolated the DRIP/TRAP protein complex. The ligand-inducible interaction between the DRIP/TRAP complex and retinoid receptors is independent of RA sensitivity and the expression of PML-RARalpha, supporting the glo

The human Cut gene, CUTL1, was mapped to chromosome 7, band 7q22, a region found in cytogenetic studies to be deleted in several human cancers. CUTL1 encodes for the human Cut protein, a homeodomain protein that was shown to bind to the promoter of the c-Myc proto-oncogene and repress its expression. As a first step in trying to determine whether CUTL1 is a tumor suppressor gene, we verified whether this gene is located within the chromosomal region that is deleted in human cancers. High resolution maps containing the CUTL1 genomic DNA were constructed in lambda phage, cosmid and PAC clones. Three (CA)n microsatellite polymorphic markers were identified within CUTL1 genomic DNA and were used in loss of heterozygosity (LOH) studies using genomic DNA from uterine leiomyomas and matched normal myometrium. LOH at 7q22 was demonstrated in 7 out 50 uterine leiomyomas samples, and the smallest commonly deleted region included CUTL1. Northern blot analysis revealed that CUTL1 mRNA levels were reduced in 8 out of 13 uterine leiomyomas. / In parallel studies, the Cut protein was found to interact with Polyomavirus large T (PyLT) protein in breast tumors and uterine leiomyomas that arise in transgenic mice expressing PyLT. Since LT was known to inactivate two tumor suppressors with which it interacts, Rb and p53, we hypothesized that genetic alterations to CUTL1 may occur in human breast cancers. Loss of heterozygosity at 7q22 was found in 18.2% (12/66) of sporadic breast tumors. The deleted region at 7q22 in every case included one or more of the three CUTL1 polymorphic markers. LOH at 7q22 was associated with increased tumor size, but not with tumor types and grades. / The exon-intron structure of CUTL1 was determined and several CUTL1 mRNA isoforms have been identified and characterized. All isoforms were found to be expressed in all tissues and tumor cells tested. Comparison of cDNA sequences with the CUTL1 genomic structure revealed a complex pattern of alternative transcription initiation, splicing and polyadenylation. Interestingly, transcription can starts in two genomic regions, and the choice between alternative polyadenylation sites and splice acceptor sites appears to be dictated by the nature of the first exon or associated promoter sequences.

Parathyroid Hormone-related Protein (PTHrP) was first identified as a tumor-derived product responsible for Malignancy Associated Hypercalcemia. The protein was characterized based on its ability to mimic Parathyroid Hormone (PTH) in kidney and bone. PTHrP is a secretory peptide with limited homology to PTH at the amino-terminus that allows it to bind to the common PTH/PTHrP receptor (PTH1R). Normal secretion of PTHrP has now been demonstrated in a number of tissues where it acts as an autocrine/paracrine mediator of cell growth and differentiation. Much of the documented biological activity of PTHrP is mediated through PTH1R however a body of evidence indicates that PTHrP uses multiple signal pathways in target tissues. One of these pathways consists of an intracellular signal mechanism that is dependent on a bipartite nucleolar targeting signal (NTS) located between residues 87--107 of the mature protein. This sequence is similar to the NTS motifs found in the nucleolar HIV-1 proteins Tat and Rev. Studies have shown that the NTS of PTHrP is both necessary and sufficient to target PTHrP to the nucleus and nucleolus of a variety of cells. Nuclear localization has been associated with altered gene expression, proliferation and inhibition of apoptosis. The mechanism used by this secretory protein to gain access to the nucleus is largely unknown. We examined the possibility that PTHrP is internalized from the cell surface and transported to the nucleolus in a manner dependent on the NTS. Using transiently transfected COS-1 cells we demonstrated NTS dependent localization of full-length PTHrP at the plasma membrane and in nuclear compartments. Site-directed mutagenesis identified an 87GxKKxxK93 motif at the N-terminus of the NTS that is critical for nuclear/nucleolar targeting. A biotin-labeled PTHrP-NTS peptide was internalized and localized to the nucleolus in a time and dose-dependent manner whereas a similarly basic NLS peptide was not. Internalization of PTHrP-N / We investigated whether binding to resident RNA structures targets PTHrP to the nucleolus. We have shown that transfected, in vitro translated and endogenous PTHrP bind to homopolymeric and cellular RNA with high affinity and specificity. Binding was dependent on the NTS and in particular the 87GxKKxxK93 motif. This motif is also a conserved sequence in double-stranded RNA binding proteins. This work represents the first demonstration of RNA binding by a secretory protein and predicts a role for PTHrP in regulating ribosome biogenesis in the nucleolus. (Abstract shortened by UMI.)

The preparation of allylsilanes by silylation of allylsulfones followed by the reductive cleavage of the silylated sulfones, is described. The use of this new methodology in the synthesis of polyenic compounds including cis and trans retinoic acid was reported. / The preparations and reactions of a series of aminomethyl substituted $ alpha$-allylsilyl anions with carbonyl electrophiles were studied. The stereoselectivity of these reactions was subject to solvents and temperature effects. For instance, the condensation of these anions with carbonyl electrophiles proceeded to give the corresponding E-homoallylic alcohols in benzene, but the same reactions at lower temperatures in the presence of dimethoxyethane leads to the formation of the Z- isomers preferentially. / A new method for the preparation of aminomethyl substituted allylsilane 1 via the hydrosilylation of a new allene was described. This allene was prepared using an improved method of elimination of $ beta$-hydroxyvinylsilanes. / A number of analogs of retinoic acid were synthesized for their use in the treatment of leukemia. Their syntheses were carried out using the Wittig reaction. Two series of these analogs showed reasonable biological activity in HL-60 and P19 screenings. A third series of compounds was inactive.(DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI)

<p> Inflammatory breast cancer (IBC) is a dreadful disease because of its rapid onset, highly invasive nature and lack of definitive treatment strategies. The underlying differences between conventional breast cancer and IBC have identified that IBC carries a unique identity, which needs to be explored and researched extensively to achieve better prognosis and disease free survival rate. Working towards the same goal, this project focuses on deciphering two unique molecular pathways that regulate IBC cell invasion and survival. </p><p> The first part of this dissertation studies the overall distribution of upregulated caveolin-1 in IBC cells <i>in vitro</i> and identifies the role of caveolin-1 in decreasing IBC cell invasion by manipulating either the expression by siRNA specific against caveolin-1, function by treatment of exogenous caveolin-1 scaffolding domain or the assembly of caveolin-1 with methyl-&beta;-cyclodextrine (M&beta;CD) treatment. Due to the scaffolding nature of caveolin-1 and previous data suggesting the involvement of RhoC GTPase in the invasive and metastatic phenotype of IBC, this project seeks to determine the co-localization of caveolin-1 and RhoC GTPase. Based on the data, I speculate that caveolin-1 regulation of RhoC GTPase activity requires presence of intermediate modulators such as Rho specific GEFs or GAPs in caveolae that ultimately cause RhoC GTPase activation and cell invasion. </p><p> The later part of this project concentrates on targeting Platelet Derived Growth Factor Receptor &alpha; (PDGFR&alpha;) by small molecule inhibitors namely, Imatinib and Crenolanib. This study is based on the earlier data that reports higher expression and signaling of PDGFR&alpha; in IBC patients when compared to non-IBC patients. The overall goals of this part of the project are to characterize PDGFR&alpha; in IBC cell lines <i>in vitro</i> and evaluate the efficacy of two receptor tyrosine kinase inhibitor molecules against PDGFR&alpha; in regulating IBC cell survival <i>in vitro</i> and <i>in vivo</i>. Intracellular localization pattern of PDGFR&alpha; and presence of PDGF ligands in SUM149 IBC cells indicated involvement of the autocrine activation mechanism. PDGFR&alpha; targeting by Crenolanib was found to affect IBC cell survival, growth, emboli formation and thus exhibits cytostatic effect on IBC tumor <i>in vivo</i>. On the other hand, Imatinib treatment over a wide concentration range failed to render any response on IBC survival. I further extended Crenolanib studies to determine if Crenolanib treatment can make IBC cells chemosensitive (reduction in the dose of current systemic chemotherapy). This was tested by treating IBC cells with Crenolanib and Docetaxel (systemic chemotherapy) either alone or in combination to evaluate the changes in cell survival. I found significant decrease in cell survival upon combination treatment of Crenolanib and Docetaxel as compared to individual treatments. Further research with respect to the mutational status and structure of PDGR&alpha; needs to be undertaken to address the possible reasons for intracellular localization of PDGR&alpha; and Imatinib resistance in IBC. </p><p> Thus, this project studies two potential biomarkers, caveolin-1 and PDGFR&alpha; that would benefit patients with early IBC detection. Understanding the role of caveolin-1 in RhoC GTPase mediated IBC invasion provides an insight into the IBC molecular pecularities. On the other hand, targeting PDGFR&alpha; by Crenolanib offers promising neoadjuvant or adjuvant chemotherapy to minimize the loco-regional spread and to achieve longer disease free survival by preventing the IBC recurrence.</p>

A thorough investigation to evaluate the biological application of quantum dots was performed. Quantum dots are novel semiconductor nanocrystals that are highly tunable in their spectral properties and exhibit strong photoluminescence and high photostability. They have been shown to be a promising optical contrast agent for biological applications, however further biological studies are needed to evaluate and characterize their applicability. In this thesis, we examined the various applications of quantum dots and leverage its optical properties for cancer imaging. First, we evaluated the effects of different nanoparticle surface coatings on the cellular uptake of quantum dots to understand quantum dot delivery into cells. Building on our knowledge of surface coating influences, we then evaluated the cytotoxicity of quantum dots, reporting new insight on the intracellular evaluation of quantum dot cytotoxicity. Next, we demonstrated the specificity of bioconjugated quantum dots in molecular targeting and imaging of cancer cell markers. Finally, we engineered a novel nanoparticle construct, an activatable quantum probe that activates in the presence of proteolytic activity as a potential method for early cancer detection based on increased metalloproteinase activity in the stroma of cancer tissue. Furthermore, we expanded our focus on developing 'smart' functional nanoprobes by developing a novel siRNA-based molecular beacon useful for gene target validation and silencing as a promising tool for dual imaging and therapy in cancer.

In 1983, Atkinson, Bartoszynski, Brown, and Thompson proposed a method of parameter estimation referred to as "simulation based estimation", or SIMEST. SIMEST is closely related to maximum likelihood, in that both methods deal with parameter estimation in the context of a fully parametric model. With SIMEST, however, the arduous step of obtaining the density function from a set of model axioms is avoided via simulation. In this dissertation, we extend the ideas of SIMEST to the case of multidimensional data. A nearest neighbor based binning scheme is proposed where the observations are divided into bins determined by the "rings" of concentric ellipsoids, the "rings" being chosen to roughly approximate regions of equal probability. The ellipsoids are each allowed to have different axes, the axes for each ellipsoid being determined by the data. Some theoretical justification is developed which establishes strong consistency for the parameter estimates obtained by this method. The theory also suggests a promising variation on the idea using many overlapping bins. Another theoretical topic related to the problems associated with global optimization in SIMEST is also treated. We explore the usefulness of these techniques in modeling the secondary tumor generation mechanisms of cancer. In one model, it is assumed that 3-dimensional data is available: (a) the time from the detection and removal of the primary to the discovery of the first secondary tumor, (b) the volume of the primary tumor, and (c) the volume of the first secondary tumor. In a second model, it is assumed that two additional dimensions of information are available (i.e. 5-dimensional data): (d) the time from the detection of the first secondary tumor to the detection of the second secondary tumor and (e) the volume of the second secondary tumor.

Metal nanoshells are a new class of optically tunable core/shell nanoparticles which are finding numerous applications in biomedicine due to their biocompatibility, chemical functionability, and optical tunability in the near infrared (a region of light where tissue is optically transmissive). The following work explores the design of near infrared resonant nanoshells for two new diagnostic and therapeutic biomedical applications. For diagnostic purposes, near infrared resonant nanoshells were found to be optically detectable in whole blood and proved capable of detecting sub-nanogram levels of analyte in whole blood in under 30 minutes. For therapeutic purposes, near infrared absorbing nanoshells were heated in tumors using higher laser powers. Excitation of near infrared resonant nanoshells promoted photothermal destruction of cancerous growths in vitro as well as in vivo. In vitro, nanoshell treated cells were photothermally ablated upon near infrared excitation with no detectable damage to cells receiving lasers only. Similar results were found in whole tissue. Using a live mouse model, a complete therapy system was demonstrated where systemically delivered nanoshells were found to preferentially accumulate into tumors. Near infrared excitation of these tumors provided complete recovery in 55% of the cases, while controls displayed a mean survival time of only 15--20 days.

Cancer remains a significant health concern today. It is the 2 nd leading cause of death in the United States. Critical to controlling cancer-associated morbidity and mortality is early detection. Early detection strategies include detecting molecular-level changes prior to phenotypic changes, enabling a sufficient amount of time for effective therapies to be implemented. Not only is early detection critical, but issues such as patient safety and cost should be considered when implementing these strategies. This thesis examines work using nanoshell-based optical contrast agents for early cancer detection using scattering-based optical imaging systems. Metal nanoshells are a novel class of optically-tunable nanoscale material that are composed of a dielectric core (usually silica) surrounded by a metallic shell (usually gold). By systematically varying the ratio between core diameter and shell thickness, the absorption and scattering maxima can be tuned to different wavelengths including those in the visible and near infrared (NIR). Specific Aim 1 addresses the fabrication of NIR scattering nanoshells for use as optical contrast agents to enable scatter-based cellular imaging. Specific Aim 2 focuses on using dual NIR absorbing/scattering nanoshells for a nanoshell-based integrated cancer imaging and therapy application. Finally, Specific Aim 3 addresses the diagnostic capabilities of gold nanoshells ex vivo using reflectance confocal microscopy (RCM).

Adhesion of malignant tumor cells under flow conditions to the endothelial monolayer lining the interior of the blood vessels is an important step in the metastatic cascade. This project examined the adhesive interactions of murine RAW117 large-cell lymphoma cells to murine hepatic sinusoidal endothelial cells (HSE). Flow cytometric analysis demonstrated constitutive-expression of VCAM-1, ICAM-1, PECAM-1 and beta1 integrin subunit on the surface of both HSE and RAW117 cells. Additionally, alpha4 and beta 7 integrin subunits and MAdCAM-1 were present on the RAW117 cell surface. The dynamic adhesion assay used employs a parallel-plate flow chamber coupled with video microscopy and digital image processing. It is capable of distinguishing initial attachment from adhesion stabilization. Using monoclonal antibodies to block the surface molecules mentioned above, we determined that an interaction of integrin alpha4beta1 on RAW117 cells with liver endothelial VCAM-1 occurs during the early stages of the adhesion process and may be important in liver metastasis. The second part of this project focused on the potential role of the cyclooxygenase (COX) pathway in the adhesive interactions of the cells studied. COX-1 isoform was detected in unstimulated RAW117 cells. Blocking it with COX inhibitors such as indomethacin, aspirin, sulindac and piroxicam resulted in increase in adhesion under static conditions. However, when lipoxygenase (LOX) inhibitors (such as ETI, NDGA and Esculetin) were used either alone or in combination with COX inhibitors, there was a significant decrease in adhesion. This observation suggested an involvement of the LOX pathway in the adhesion process. COX-2 isoform was detected in unstimulated HSE and was upregulated after stimulation with TNF-alpha. Blocking it in unstimulated HSE resulted in an increase in adhesion of RAW117. However, in the case of TNF-alpha stimulated HSE, NS-398 (a COX-2 specific inhibitor) caused a significant decrease in adhesion, indicating a potential role for COX-2 in the adhesion of RAW117 to stimulated HSE.