Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: A, page: . Adviser: David Tewksbury. Includes bibliographical references (leaves 132-149) Available on microfilm from Pro Quest Information and Learning.

<p> <b>Statement:</b> A global <i>Rbp1</i> knock out (<i> Rbp1-/-</i>) mouse model was used to correlate direct retinoid measurements with vitamin A metabolizing and atRA biosynthesizing enzyme activities, Crbp function and tissue microenvironment for the first time. </p><p> <b>Methods:</b> atRA was quantified by LC-MRM³ and ROL/RE/RAL was quantified by HPLC-UV. Enzyme activities were measured from enzymes present in subcellular fractions isolated from WT and <i>Rbp1-/- </i> tissues. Mouse CrbpI and CrbpIII were purified from transformed <i> Escherichia coli</i> for functional comparative studies. Tissue were formalin fixed for histological examination. Relative gene expression was analyzed using quantitative PCR. </p><p> <b>Results:</b> Reduced atRA was consistently quantified in extrahepatic tissues with elevated ROL/RE. Relative gene expression showed altered expression in retinoid pathway proteins and atRA loss preceded expression changes in some cases. Tissue microenvironments also consistently showed a loss of structure and organization along with accumulation of extracellular matrix and hyperplasia without apparent disease. Functional studies showed that CrbpIII binds retinol with less affinity than CrbpI and does not function equivalently to CrbpI in regulation of atRA biosynthesis. Also, metabolizing enzymes had altered activities in the <i>Rbp1-/-</i> tissues with reduced atRA biosynthesis. </p><p> <b>Conclusions:</b> Loss of CrbpI results in altered regulation of enzyme activity and atRA homeostasis cannot be maintained by other Crbp homologs in extrahepatic tissues. Dysfunctional atRA biosynthesis due to loss of CrbpI results in altered tissue microenvironment characteristic of dietary vitamin A deficiency and precancerous dysfunction associated with cancers that are observed to have silenced CrbpI.</p>

Exposures to a variety of agents, including the tumor-promoting agent 12-O-Tetradecanoylphorbol-13-acetate (TPA), elicit inflammatory responses, which culminate with the release of reactive oxygen species (ROS) by phagocytes. Current hypotheses regarding tumor promotion include that ROS produced by phagocytic and epithelial cells may cause alterations in proliferation and/or differentiation of initiated cells within promoted tissues. Therefore, ROS production in response to TPA was investigated in HL60 phagocytes and MCF-7 mammary epithelial cells. The exposure of HL60 cells to TPA (0.1 μM) induced superoxide production and accumulation of gp91phox mRNA, which encodes for the superoxide-producing subunit of NADPH oxidase. Inhibiting transcription indicated TPA did not increase gp91phox mRNA stability. Transient transfection assays revealed that TPA-enhanced luciferase activity driven from a gp91phox promoter-reporter (p91phox). Inhibition of Activator Protein-1 (AP-1) activity by a transactivation mutant (TAM67) variant of c-Jun abrogated the TPA-responsiveness of p91phox. DNA binding assays with oligonucleotides derived from the gp91phox promoter led to the identification of a non-consensus AP-1 binding sequence (5'-TGAGTAA-3 ' = phox-TRE) located at -1090 of gp91phox. Members of the Jun and Fos families contributed to the formation of an AP-1 complex at the phox-TRE. Furthermore, point mutations in the phox-TRE abrogated AP-1 binding and the responsiveness of the 1.5-kb gp91phox promoter to TPA. In MCF-7 cells, TPA (0.1 μM) induced oxidation of the intracellular probe, 2', 7'-dichlorofluorescin-diacetate (DCFH-DA), an indication of an increased oxidative state. Since gp91phox mRNA was not detected in MCF-7 cells in the presence or absence of TPA, ROS emitted by gp91phox were ruled out as the source of oxidation. Due to its known peroxidase activity, the TPA-inducible cyclooxygenase-2 (COX-2) enzyme was investigated as the source of the increased DCFH-DA oxidation induced by TPA. Data supported that DCFH and DCFH-DA detected COX-2 activity in enzymatic and cellular assays, respectively. In vitro assays using recombinant human COX-2 yielded increased DCFH oxidation in the presence and absence of arachidonic acid. In cellular assays, the time-dependent TPA-induced DCFH-DA oxidation paralleled COX-2 expression and activity induced by TPA. Furthermore, the COX-2 specific inhibitor NS398 (50 and 100 muM) inhibited the TPA-induced oxidation of DCFH-DA in MCF-7 cells. COX-1 and -2 inhibitors, indomethacin and ibuprofen provided similar effects, but sulindac was less effective. In conclusion, gp91phox was regulated transcriptionally in response to TPA and the phox-TRE, which interacted with AP-1, was required for TPA-induced gp91phox promoter activity. These data support that increased ROS levels in TPA-promoted tissues may be due to ROS production by phagocytes. Additionally, the production of ROS by phagocytes may be mediated by AP-1 dependent up-regulation of gp91phox. The increased oxidative state detected in MCF-7 epithelial cells following TPA exposure was attributed to increased COX-2 activity. Therefore, DCFH-DA may be useful to screen for drugs that may have potential to induce or inhibit COX-2.

Intracellular drug accumulation was studied in two drug resistant variants of the human breast cancer MCF-7 (MCF/7) cell line selected with mitoxantrone (MCF7/Mitox) and doxorubicin (MCF7/D40). Earlier studies show that both cell lines have similar cell cycle characteristics, and both are multidrug resistant. Previously, P-glycoprotein was detected in MCF7/D40, but not in MCF7/Mitox. Both cell lines, however, display decreased drug accumulation. The P-glycoprotein chemo-modulator verapamil increased mitoxantrone accumulation 1.6 fold in MCF7/D40 cells, thus achieving identical intracellular drug levels to the MCF7/S cell line. Verapamil had little effect on drug accumulation in MCF7/Mitox cells. Rapid influx of mitoxantrone from 5 seconds to 60 seconds was not significantly different between MCF7/Mitox and MCF7/S. Influx in the MCF7/D40 cell line was greater than in the MCF7/Mitox or MCF7/S cell lines. Decreased drug accumulation was found to be at least partly due to enhanced drug efflux. Depletion of 73.9% to 88.9% of cellular ATP by sodium azide (NaN3) decreased the efflux of mitoxantrone in each cell line, thus demonstrating an energy dependence of drug efflux.

The UVB portion (280--320 nm) of the ultraviolet spectrum contributes to the development of non-melanoma skin cancer (NMSC) in humans. UVB irradiation causes epigenetic alterations in target keratinocytes, including the upregulation of Activator Protein-1, a transcription factor complex that alters normal cellular gene expression. c-Fos expression is induced in a manner that correlates with the UVB-induced activation of AP-1. This suggests that c-Fos functions as a major regulatory component in the UVB-induced transactivation of AP-1. The purpose of this dissertation is to characterize UVB-induced regulation of c-Fos expression. Transcriptional regulation of c-fos is investigated by evaluating the role of each of four cis elements within the c-fos promoter. While mutation of each of these four cis elements results in significantly lower levels of UVB-induced promoter activity, the CRE and FAP1 elements mediate most of the UVB transactivation of c-fos. In addition, UVB irradiation induces homodimers of phosphorylated CREB to bind to the CRE and FAP1 elements. To identify cellular signal transduction pathways that are induced by UVB-irradiation to regulate c-Fos expression, a UVB-inducible enzyme, phosphatidylinositol 3-kinase (PI 3-kinase), is studied. Inhibition of PI 3-kinase reduces c-Fos expression in UVB-irradiated cells. Akt and GSK-3beta, constituents of the PI 3-kinase signaling pathway, are also found to be part of this UVB-induced signaling pathway. To identify potential molecular targets for the development of skin cancer chemoprevention strategies, the polyphenolic compound nordihydroguaiaretic acid is tested and found to prevent UVB-induced c-Fos expression and AP-1 transactivation by inhibiting the PI 3-kinase signal transduction pathway. Thus, phospho-CREB binding to the CRE and FAP1 cis elements and PI 3-kinase signaling are both identified as molecular mechanisms and potential molecular targets that are involved in UVB-induced c-Fos expression and AP-1 transactivation.

Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, the human colon tumor cell line HT29 (wtAPC -/-) was stably transfected with a zinc-inducible wild-type APC gene. Addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein, and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase (betaGal) gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. To examine the role of APC-dependent regulation of ODC, the two sets of E-boxes were analyzed. When the E-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results indicate that APC expression can inhibit ODC via the 5' E-box. Using the cell model previously described, APC selectively represses the ODC A allele, apparently through selective binding of Mad1. These results demonstrate that wild-type APC suppresses c-myc and activates Mad1 expression in HT29 colon-derived cells. Treatment of Min mice with the ODC inhibitor, difluoromethylornithine (DFMO), suppresses intestinal polyamine contents and intestinal tumorigenesis. The data presented in this dissertation indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. Apoptosis is significantly reduced in both the small intestines and colons of Min (multiple intestinal neoplasia) mice when compared to normal littermates. Apoptotic indices can be restored by treating the mice with alpha-difluoromethylornithine (DFMO). DFMO is a specific, irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. These results indicate that APC induces apoptosis via the mitochondrial pathway rather than through the death receptor pathway. APC also affects a variety of other proteins involved in the regulation of apoptosis including transcription factors (i.e., ets2, FKHR, JunB, etc.) and bcl-2 (i.e., Bcl-xL) family members. The multiple levels at which APC functions suggest a variety of possible targets for the prevention and treatment of colon cancer. (Abstract shortened by UMI.)

Purpose. Explore how nurses and patients talk about cancer pain management during an oncology clinic visit. Describe the elements of these interactions and the patient-researcher discussions in order to evaluate the communication process used to report pain and to plan pain management. Design. Exploratory design of nurse-patient oncology clinic interactions and patient-researcher discussions. Methods. Nurse participants completed (1) a questionnaire about clinic time spent with patients and (2) Ward's Barrier Questionnaire (BQ) which concerns beliefs about the use of analgesics. Patient participants also completed a questionnaire about pain and Ward's BQ. Nurse-patient clinical interactions were audio-taped and analyzed by means of narrative analysis. Post-questionnaire patient-researcher discussions were analyzed also by narrative analysis. Quantitative data analysis was conducted on data from the questionnaires. Findings. Audio-taped nurse-patient interactions were divided by theme grouping into four summary examples: (1) Beginning to want to put it all together (56%), Communicating personal uniqueness (22%), (2) Active patient participation (13%), and (3) Learning about tests for future treatment (9%). Analysis revealed that while over 60% of the participants reported to be presently in pain, pain and pain management were rarely mentioned during the interactions. Patients did talk about pain extensively during the post-questionnaire discussions. Conclusions. Narrative analysis of nurse-patient interactions can provide health care professionals with examples of the quality and extent of information that cancer patients need regarding pain management. Not enough attention is given to patients' pain reports in the planning of pain management. Without systematic study of patients' pain reports and patients' comments on the effectiveness of analgesics, oncology clinic pain management will continue to remain inadequate.

Choline metabolites are elevated in breast cancer, decrease in response to effective therapy and are detected non-invasively by magnetic resonance modalities. Decreases in choline metabolites occur early-on after initiation of treatment. There is potential for use of choline metabolites as non-invasive diagnostic and therapeutic response indicators. Choline metabolites are detected in vivo by magnetic resonance spectroscopy (MRS) in broad resonances which are composites of multiple compounds. Tumor extract studies have suggested that phosphocholine (PCho) is the component of these resonances with the greatest potential for use as a diagnostic marker or therapeutic response indicator. Since other compounds present in these broad resonances vary in concentration with cancer progression and in response to therapy, changes in these other resonances can potentially diminish the overall signal or dampen the detectable therapeutic response. The ability to resolve and quantify PCho in vivo increases the sensitivity of this detection method, and hence, increases its potential utility. Herein is reported the in vivo resolution and quantification of PCho in a human breast cancer xenograft model in mice. A significant PCho decrease is detected following treatment with the taxane docetaxel. This PCho decrease is correlated with the diffusion-weighted magnetic resonance imaging (DWMRI) measured increase in tumor water mobility, and with mitotic catastrophe, a non-apoptotic mode of cell death. By studying model system of human breast cancer cells, other metabolites in the choline pathway varying with cancer progression are determined, and the transcriptional expression of genes in the choline pathway is quantified. From these data and enzyme activity data reported by other groups, a model is proposed where a number of metabolic perturbations combine to elevate PCho in breast cancer. These perturbations include the elevation of choline transporter, choline kinase, and phospholipase activities, in combination with decreased CTP:PCho cytidylyltransferase (CCT) activity. By changes in metabolites and gene expression following therapy, it is proposed that increased CCT activity combined with decreased phospholipase and GPC phosphodiesterase activity lead to decreased PCho. In addition, expression of a putative choline transporter (CTL1 variant A) and a putative choline kinase (CHKL) is quantified in human breast cells.

The cellular infiltration of solid tumors is indicative of an immune response to cancerous growths. Unfortunately, most tumors grow progressively despite this infiltration. Therefore, the infiltrate from a regressing tumor is necessary in order to examine the requirements for tumor rejection. Due to the rarity of tumor rejection, elucidating the requirements is difficult without an animal model. The sponge model of concomitant tumor immunity allowed the examination of the components associated with tumor rejection. In the model of concomitant tumor immunity an animal is given a primary tumor followed by a secondary tumor challenge. Despite the progression of the primary tumor, the secondary tumor challenge is rejected. In this model the secondary tumor challenge is delivered into a preimplanted gelatin sponge matrix which can be retrieved in order to capture the components associated with tumor rejection. Retrieval of both the primary progressing tumor and the gelatin sponge allowed a direct comparison of the factors associated with tumor progression and rejection. Using this model, we have examined the progressing and rejected tumor sites for differences in T cell cytotoxicity, V beta T cell receptor usage, and the expression of cytokine genes and signal transducing proteins. The results from this study demonstrated that the T cells isolated from progressing tumor sites were not cytolytic, whereas the T cells from the rejection sites showed significant cytolysis towards the autologous tumor cells in vitro. Surprisingly, the T cell infiltration into the progressing and rejected tumor sites were similar with V beta 1 and V beta 8 T cell receptor bearing T cells predominating at both locations. The T cell response also showed clonal restriction upon examination of the complementarity determining region 3 (CDR3) of the T cell receptor. Significantly, the rejection site showed higher gene expression levels of IFN-γ, TNF-α, IL-2, IL-4, IL-10, and IL-12 and reduced TGF-β gene expression compared to the progressing tumor site. Finally, although the T cells from the progressing tumor site showed an altered pattern of tyrosine phosphorylation, the signaling molecules p59ᶠʸⁿ and CD3 ζ were expressed at comparable levels in the T cells from both sites. These data strongly suggest that the tumor microenvironment may play a major role in orchestrating an anti-tumor immune response.

The ability of specific cytokines to increase the immunogenicity of poorly or non immunogenic tumors is of extremely important clinical value because it can be exploited in the generation of an effective antitumor vaccine as a potential modality for treating cancer. In this scenario, the patient's tumor cells are transfected with the cytokine gene of interest, irradiated and re-injected into the patient as a vaccine with the potential of eliminating tumor recurrence or metastatic foci. Interferon-γ (IFNγ) is an important cytokine whose immunomodulatory properties include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. Therefore IFNγ represents one of the most suitable candidates for a cytokine-transfected-tumor vaccine approach. In this study a retroviral vector was used to introduce the IFNγ gene into a murine mammary carcinoma cell line (EMT6), poorly responsive to exogenous IFNγ stimulation, to assess the effect of IFNγ transgene expression on tumor immunogenicity. Transductants expressed the IFNγ transgene, class II MHC antigens and secreted IFNγ. The induction of class 11 MHC in IFNγ-transduced cells correlated with expression of a mouse class II transactivator (CIITA), a cytoplasmic protein involved in the activation pathway of class II MHC gene. Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was inhibited in immunocompetent mice. Similar findings were demonstrated using an IFNγ gene-transduced melanoma cell line. Rechallenge of animals that rejected an IFNγ-transduced EMT6 clone with parental EMT6 cells resulted in tumor rejection, suggesting that IFNγ-transduced EMT6 cells were able to induce long term immunity. Vaccination of animals with low dose cytokine-transduced cells induced partial or complete protection against rechallenge with parental cells suggesting that significant clinical benefit may be achieved using a small fraction of cytokine-transduced cells as a vaccine. In addition, these studies demonstrated that the immunogenicity of tumor cells poorly responsive to exogenous IFNγ can be enhanced by inserting and expressing the IFNγ transgene. IFNγ-transduced cells expressing class 11 MHC may function as antigen by presenting cells presenting tumor associated antigens, suggesting a role for class II MHC in reducing EMT6 tumorigenicity and inducing long term tumor immunity.