The process of colon carcinogenesis is dependent upon a variety of genetic mutations and intestinal luminal risk factors. These risk factors cooperate to influence pathophysiological targets directly linked to the carcinogenic process. Cyclooxygenase-2 (COX-2) is one such target, and it is found to be upregulated in 50% of colonic adenomas and 85% of carcinomas. The interaction between genetic mutations and luminal risk factors to affect COX-2 expression was explored in this dissertation work. An activated K-ras oncogene and mutant APC tumor suppressor gene both regulate COX-2 posttranscriptionally. Using colon cancer cells with these genetic alterations, we determined that an activated K-ras stabilizes COX-2 mRNA and works through a PKC-dependent mechanism in a cell line-specific manner. K-ras also affected PTEN expression to potentially suppress cell survival. Wild-type APC downregulated COX-2 protein levels without affecting mRNA. APC interfered with PI-3K-dependent signaling of COX-2, and affected PKC-depending signaling to further modulate COX-2 expression. In addition to genetic alterations, luminal factors obtained from the diet influenced COX-2 expression in these colon cancer cell models. Secondary bile acids like deoxycholic acid (DCA) mediated COX-2 protein stability in addition to its known affects on transcription. DCA's affects on COX-2 protein were partially mediated by APC mutational status and PKC-signaling. Polyamines, another luminal risk factor for colon cancer, regulated COX-2 through the induction of RNA processing by the polyamine-dependent gene, eIF-5A and through a reliance on transport through the cell membrane. The chemopreventive agent, Sulindac, has two metabolites---sulindac sulfone and sulindac sulfide. Previous to this study, little was known about the mechanism of action of the sulfone metabolite. We determined that sulindac sulfone suppressed COX-2 protein levels, thus impinging on this protein's ability to signal downstream genes or to produce prostaglandins. In addition, DFMO, a chemopreventive agent that suppresses polyamine synthesis, significantly induced COX-2 protein levels, predicating the importance of combination chemoprevention to battle COX-2-driven tumorigenesis. The intricate cooperation of genetic mutations and luminal risk factors affecting COX-2 expression and subsequently tumorigenesis, suggest that multiagent interventions may be necessary for successful strategies of colon cancer chemoprevention.

Considerable changes in the practice of chemotherapy have occurred which include the shift of the majority of therapy to an outpatient setting and the availability of more effective antiemetic agents to treat nausea and vomiting. The outpatient delivery of chemotherapy is also characterized by limited nutrition intervention. Data are not available that define the impact of these changes in chemotherapy practice on the nutritional status of outpatients receiving chemotherapy. Consequently, this study was designed to address four primary aims. The first aim was to observe the change in degree of common side effects, or symptom distress, and in nutritional and functional status measures over 3 months in outpatient chemotherapy patients. The second aim was to test the utility of two versions of a nutrition risk scoring tool. Version A includes weight change and subjective appetite ratings. Version B incorporates change in fat-free mass (FFM) measures by bioelectrical impedance analysis (BIA) and energy and protein intakes. The third purpose was to examine the relationship of the nutritional and other factors studied with response to chemotherapy. Finally, comparisons were made among the body composition results from skinfold thickness measures and BIA using instrument manufacturer-supplied and population-specific equations. A key finding from this study was that Nutrition Risk Score A detected nutritional change over time and lower scores were correlated with positive chemotherapy response. Unanticipated findings from this study were the significant gender differences in nutritional and clinical factors and their relationships to chemotherapy response. Male subjects experienced weight loss and an overall decrease in nutritional status as demonstrated by an increase in Nutritional Risk Score A, while female subjects did not have weight loss and had a trend towards improved Nutritional Risk Score A. The Kushner et al (1992) BIA equation produced the closest estimates of body fat mass to those obtained using the Durnin and Womersley (1974) skinfold method. The population-based BIA equations were not interchangeable with each other or with the manufacturer's equation. Except for female breast cancer patients, the population-based BIA equations were interchangeable with each other for estimating FFM, but not with the manufacturer's equation.

Many cancer cells develop resistance to apoptosis. It is important to understand how this phenotype develops, so that these cancers can be effectively treated. The results presented in this dissertation describe two molecular mechanisms of apoptosis resistance. A mouse keratinocyte model system consisting of the benign 308 parental cell line and two malignantly progressed variants (6M90 and 6R90 cells) were used to explore the relationship between reactive oxygen species (ROS) and apoptosis resistance. Previous work showed elevated basal levels of ROS in 6M90 and 6R90 cells. The results shown here demonstrate increased resistance to UV-induced apoptosis of the variants compared to the parental line. Pharmacological and genetic approaches were used to decrease the steady-state levels of ROS in the two malignant cell lines. This increased their sensitivity to apoptosis. ROS are implicated in the activation of the anti-apoptotic Akt kinase. 6M90 and 6R90 cells had higher levels of activated Akt. Modulation of ROS levels in the 6M90 and 6R90 cells decreased the levels of activated Akt. These studies provide a molecular mechanism to explain the chronically elevated ROS and apoptosis resistance seen in many tumors. Another mechanism by which tumor cells resist apoptosis is to upregulate the anti-apoptotic protein Bcl-2. A putative response element (PPRE) for the peroxisome proliferator activated nuclear receptor (PPAR) was found in the 3' UTR of bcl-2. Further experiments indicated that the gamma subtype of PPAR bound the putative PPRE and could activate transcription. In cells transfected with PPARgamma, increased levels of bcl-2 mRNA and Bcl-2 protein were seen as compared to empty vector-transfected cells. When treated with bile acids to induce apoptosis, the PPARgamma-transfected cells were twice as resistant as empty vector-transfected cells. These studies show, for the first time, that a sequence within the 3' end of the bcl-2 gene can regulate transcription of the gene through interactions with PPARgamma. These findings may be particularly relevant in colon cancer, where PPARgamma and Bcl-2 are often overexpressed.

The ability of dendritic cells (DCs) to stimulate tumor-specific T lymphocyte responses has made them prime candidates for cancer immunotherapy. Numerous studies have focused on enhancing the ability of DCs to stimulate the immune response. Commonly overlooked is the immunosuppressive milieu of the tumor microenvironment. Many tumor types including the murine mammary carcinoma cell line 4T1, produce large quantities of the immunosuppressive cytokine transforming growth factor-beta (TGF-β). These studies have determined that TGF-β suppresses numerous dendritic cell functions involved in generating an effective anti-tumor immune response. By reducing the amount of TGF-β present in the tumor-bearing host the migratory ability and anti-tumor activity of DCs are enhanced. Adenovirus-mediated Smad7 gene transfer, was able to reduce the responsiveness of DCs to TGF-β in vitro. Vaccination of tumor-bearing mice with AdSmad7-infected DCs resulted in enhanced IFN-γ secretion by tumor draining lymph node (TDLN) cells. However, this did not translate to improved ant-tumor activity. These results stress the necessity to eliminate tumor-derived immunosuppressive molecules such as TGF-β in order to improve the effectiveness of DC-based vaccines in treating cancer.

Chronic myelogenous leukemia (CML) is a clonal disorder characterized by proliferation of cells expressing BCR-ABL fusion proteins. The BCR-ABL fusion proteins are tumor-specific antigens and represent reasonable targets for immunologic approaches against CML. We have utilized a free-solution-isoelectric focusing technique (FS-IEF) to obtain chaperone rich cell lysates (CRCL) from tumors. We found that CRCL derived from 12B1, an aggressive bcr-abl⁺ murine tumor activated dendritic cells (DCs) by upregulating CD40 and MHC-II on their cell surface and stimulating them to produce interleukin-12 (IL-12). Vaccination of mice with 12B1 CRCL pulsed DCs generated potent, tumor specific, long lasting, CD4⁺ and CD8⁺ T cell dependent immune responses. We have further demonstrated that immunization with 12B1 CRCL induced BCR-ABL specific cytotoxic T lymphocytes, indicating that BCR-ABL peptides are chaperoned by CRCL and are cross-presented to CD8⁺ T cells. Moreover, other antigenic peptides may also be present in the antigen repertoire of CRCL and contribute to the superior immunogenicity of 12B1-derived CRCL. To better optimize immunity against CML, we investigated the effects of combining imatinib mesylate with 12B1 CRCL. Imatinib mesylate specifically inhibits BCR-ABL kinase activity and has been very successful in treating patients with CML. However, the development of drug resistance has led to drug combination approaches. We have shown that the combination of imatinib with DCs loaded with 12B1-derived CRCL yielded potent anti-tumor activity. In addition to tumor-derived heat shock proteins, we have also studied the immunogenicity of apoptotic leukemic cells. We have previously reported that vaccination of mice with stressed apoptotic leukemic cells elicited potent anti-tumor immunity. We have found that stressed apoptotic leukemic cells, compared with non-stressed apoptotic ones, have higher capacity to upregulate CD40, CD80, and CD86 on the surface of DCs, to stimulate DCs to secrete IL-12, and to enhance their immunostimulatory functions in a mixed lymphocyte reaction (MLR). These findings demonstrate that apoptotic tumor cells can be immunogenic when stressed, and that DCs play a key role in determining whether a T cell response will be generated.

Epidemiological data suggest that non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumorigenic activities against colorectal cancer. NSAIDs work by inhibiting cyclooxygenases (COX) enzyme. Sulindac, a NSAID prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Microarray analysis was used to detect COX independent effects of sulindac on gene expression in human colorectal cells. Spermidine/spermine N 1-acetyl transferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was one of the genes induced by clinically relevant sulindac sulfone concentrations. Promoter analysis and mutational studies were done to map the sulindac sulfone dependent response sequences in SSAT 5' flanking sequences, which led to the identification of two Peroxisome Proliferator Activated Receptors (PPARs) response elements (PPREs) in the SSAT gene. PPRE-2 is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPARgamma in the Caco-2 cells, while PPRE-1 is not required for the induction of SSAT by sulindac sulfone, but can be bound by both PPARdelta and PPARgamma. Clinically relevant concentrations of sulfone reduced intracellular polyamine levels, inhibited cell growth and induced apoptosis in colon cancer cells. Further, only sulindac sulfone induced apoptosis could be partially rescued by exogenous polyamines. Upon evaluating other NSAIDs for their action on SSAT gene expression, it was found that they induce SSAT mRNA in either a COX dependent or independent mechanism in colon cancer cells. Studies with physiologically relevant concentrations of aspirin show that these concentrations can induce SSAT expression thereby leading to a decrease in polyamine levels. Activating mutations in K-ras, which is a late process in colon carcinogenesis, led to the suppression of SSAT expression in the Caco-2 cells due to the inhibition of PPARgamma by ERK. K-ras didn't have any effect on the induction of SSAT by sulindac sulfone but partially abolished the apoptosis caused by sulindac sulfone, indicating a possible role of mutant K-ras in sulindac resistant colon polyps. Sulindac sulfone, or Exisulin(TM) have been recently used in clinical trials for the prevention of colon, lung and prostate cancer. The data shown here, suggest that one of the mechanisms, by which sulindac sulfone could act as a chemopreventive agent is to induce the expression of SSAT thereby leading to a decrease in the intracellular polyamines. This reduction in polyamines plays an important part in the apoptosis induced by sulindac sulfone in the colon cancer cells. Further, induction of SSAT seems to a general mechanism for different NSAIDs like aspirin, indomethacin, ibuprofen, sulindac and celecoxib in colon cancer. Aspirin is able to induce SSAT and decrease intracellular polyamines at physiological concentrations, which can lead to a significant reduction in adenoma recurrence. Also, activated K- ras suppressed SSAT, but was not able to abolish the induction of SSAT by sulindac sulfone indicating the potential of using sulindac sulfone in colon cancer chemoprevention.

The estrogen receptor (ER) plays a major role in breast cancer progression, and ER+ tumors respond favorably to hormonal manipulation. The selective estrogen receptor modulator (SERM) tamoxifen (Tam) induces remissions in most ER+ patients. However, acquired resistance is often observed. Tam-resistant breast cancer is sensitive to other antiestrogenic compounds, but resistance to these agents has also been described, illustrating a major limitation to antiestrogen therapy. Therefore, we investigated a ligand-independent approach for treating Tam-resistant breast cancer by targeting the molecular chaperone Hsp90. The ER exists in a multi-protein complex containing Hsp90, which regulates the activity and stability of the receptor. Hsp90 regulates the stability of other proteins relevant to breast cancer, including Akt and Raf-1. The benzoquinone ansamycin antibiotic geldanamycin (GA) and its clinically relevant analog, 17-demethoxy-17-allylaminogeldanamycin (17AAG), bind to Hsp90 and induce the degradation of Hsp90 clients. In these studies, we show that GA depletes ER levels in Tam-resistant cell lines, and the Hsp90 clients Akt and Raf-1. Unexpectedly, Tam inhibited GA-induced degradation of the ER, but not Akt and Raf-1. This effect was consistent in vivo, where ER levels were measured in tumor xenografts growing in Tam-supplemented mice. However, Tam-stimulated tumor growth was inhibited by 17AAG, and tumor Akt and Raf-1 levels were downregulated. Immunoprecipitation experiments showed that Tam does not inhibit GA-induced changes in the ER-chaperone complex, suggesting an alternate mechanism for the inhibition of GA-mediatied ER degradation. Through cell fractionation, immunostaining, and chromatin immunoprecipitation experiments, we have found that the mechanism involves prolonged association of the ER with the DNA in the presence of Tam, which leads to nuclear accumulation of the ER and sequestration from the proteasome. Furthermore, inhibition of GA-induced ER degradation was inhibited by another SERM, Raloxifene, indicating that the effect is not Tam-specific. Based on its ability to downregulate critical signaling proteins involved in breast cancer, including the ER, 17AAG may provide a useful alternative for patients that have failed hormonal therapy. Because SERMs inhibit the degradation of ER protein induced by GA, they may compromise the efficacy of GA on ER activity, and combined therapy should be approached with caution.

Tumor specific antigens are the holy grails of cancer research. We made an attempt to identify novel tumor antigens by examining the tumor infiltrating B lymphocyte (TIL-B), and lymph node derived B cell (LN-B) population. TIL-B and LN-B cells were tested for reactivity against glutaraldehyde-fixed allogeneic cell lines by enzyme linked immunosorbant assay (ELISA). Of the 466 wells containing TIL-B cells, 29 (6.2%) of them produced antibodies that were reactive to allogeneic tumor cell lines. Of the 712 wells containing LN-B cells, 79 (11.1%) of them produced reactive antibodies to allogeneic tumor cell lines. Unfortunately, we were unable to identify specific clones producing those reactive antibodies by limiting dilution. Furthermore, we examined the murine immune response to a truncated p53 harboring four point mutations and fused to enhanced green fluorescent protein (EGFP). Balb/c mice were immunized with either plasmid DNA or the recombinant protein. We found that delivery by intramuscular injection of plasmid DNA encoding truncated, mutant p53 along with murine GM-CSF mice resulted in non-measurable antibody response and minimal cellular cytotoxic activity while recombinant protein immunization resulted in a strong antibody response and no measurable cytotoxic activity. We conclude that immunization with the fusion construct containing green fluorescent protein does not enhance cytotoxic responses to mutant p53. In addition, the creation of multiple point mutations in the p53 gene may have prevented the fusion protein from being processed and expressed on MHC class I molecules.

<p> The purpose of the study was to examine the impact of cancer caregiving on primary caregivers, exploring their personal narratives looking back on the entire experience from diagnosis, through treatment, and beyond. Caregiving is associated with exacerbation of stress-related disorders such as hypertension and heart disease and may also be associated with increased mortality rates. Transitions theory served as the conceptual framework for the study. Eleven adult caregivers, pre-retirement age, each participated in two semi-structured interviews. Caregivers were recruited from a community cancer resource center and were purposively selected to achieve maximum variation in terms of outcome of cancer treatment. The sample included 8 females and 3 males; there were 3 husbands, 6 wives, and 2 daughters. Caregivers provided care for patients with a variety of cancer types and a variety of treatment outcomes, from cancer free with sequelae to deceased. Each caregiver interview recording was transcribed, and preliminary examination of each transcript helped guide subsequent interviews. NVivo9 software was used to assist with data management. Data saturation was achieved. Narrative within-case analyses as well as thematic analysis were used to address research questions. Thematic analysis resulted in seven themes: Burden: The Load that Never Ends; Disconnectedness and Isolation: The Invisible Person; Helplessness and Loss of Control: Tied to This Ride; Dealing with the Healthcare System; Role Disruption: Spinning the Plates; Loss, Change, and Grief: Reaction to the Whole; and Carrying Forward with Scars: New Priorities and Permanent Change. All of the caregivers changed their employment or social responsibilities due to the demands of caregiving. Themes were present in different parts of the cancer trajectory and in differing intensities in all interviews. Findings included disconnectedness and isolation as a central feature of cancer caregiving, plus significant grief present through the cancer trajectory, especially in the post-treatment phase. Furthermore, the experience of cancer caregiving remained one of significant impact years after treatment had ended. Successful transitioning requires connectedness and mastery, but participants in this study identified that their caregiving trajectories were full of isolation, grief, burden, and helplessness. Many suggested the need for support, even though they tended to deny their own physical and emotional needs while caregiving. Healthcare professionals can help by providing information, support, listening, and grief counselling. Research is needed on interventions that may reduce isolation, helplessness, and burden for caregivers.</p>

The effect of CD40, CD54/ICAM-1 and CD95 (Fas/APO-1) ligation in murine B lymphomas was investigated. Crosslinking ligation of CD40 induced p53, the p53-regulated CDKI p21Cip1/Waf1, and apoptosis in 3 lymphoma cell lines of a mature phenotype (A20, M12 and TA3), but not in 2 lymphomas of an immature phenotype (WEHI-279 and WEHI-231). Association of Mdm2 with p53 was reduced in A20. Expression of Bax and Bcl-2 was unaffected by CD40 ligation in all lines. Ligation of CD95 induced apoptosis in A20, not in M12, TA3 or WEHI-279. Crosslinking ICAM-1 on A20 had no effect on growth, but induced tyrosine phosphorylation of a 90--100KDa band in ICAM-1 immunoprecipitates, consistent with ICAM-1 itself. My results demonstrate that CD40 and CD95 differentially affect B cell lymphomas according to their developmental stage and show the potential for tumor suppression by signals that promote growth of non-transformed B cells.