Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 147 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 135 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph.D. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2009.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references (leaves 203-234).

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Bibliography: leaves 166-195.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 148-164). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Cancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.

Chaperone proteins such as heat-shock proteins 70, 90 and 110, glucose-related protein 94 and calreticulin have been reported to be effective anti-tumor vaccines when purified from a tumor source. We have developed a procedure utilizing a free-solution-isoelectric focusing technique to obtain vaccines from tumor or normal tissue sources that are rich in multiple immunogenic chaperone proteins, called Chaperone-Rich Cell Lysate (CRCL). Tumor-associated peptides are presumed to be the currency of T-cell mediated anti-cancer immunity, and tumor-derived chaperone vaccines are believed to be purveyors of such peptides. As a novel anti-cancer strategy, we have examined the extent to which the peptide repertoire of CRCL can be manipulated. Here, we explored the concept of creating a designer CRCL, utilizing the adjuvant properties and the carrying capacity of CRCL to deliver exogenous antigenic peptides for DC-based presentation and ultimately demonstrate the anti-tumor efficacy of the designer vaccine in vivo. Designer CRCL allows for the development of personalized vaccines to those afflicted with cancer expressing known antigens.Growing evidence indicates that the stress response, specifically involving HSPs, has a profound impact on tumor immunogenicity. Enhancement of T-cell-mediated immunogenicity correlates with the expression of inducible heat shock protein 70 (iHSP70), the major heat-inducible member of the HSP70 family. In addition, studies have shown tumor-specific cell surface localization of iHSP70 correlates with an increased sensitivity to lysis mediated by human natural killer (NK) cells. Given these findings, investigating novel and effective means of modulating the heat shock response within tumor cells may bear great therapeutic potential and result in potent anti-tumor immune activity. Withaferin A (WA) is a compound isolated from the plant Withania somnifera that has been shown to induce a robust transcriptional heat shock response. In our studies, we found that WA treatment resulted in increased surface expression of iHSP70 in several tumor types leading to significant immunostimulatory effects. These findings indicated that WA-dependent modulation of the heat shock response may enhance tumor immunogenicity. Given the potent immunomodulatory and anti-tumor effects of WA as well as the adjuvanticity and specificity of peptide-complexed CRCL against tumors, these therapies individually have shown profound anti-cancer activity.

In eukaryotes, the ubiquitin-proteasome system (UPS) degrades most cellular protein. Inhibition of the UPS has been associated with different disease states and can affect various intracellular processes including the activation of heat shock protein (hsp) gene expression. During cellular stress, HSPs act as molecular chaperones by inhibiting protein aggregation and assisting in their refolding once normal conditions are re-established. In the present study, Withaferin A (WA), a steroidal lactone with possible anti-inflammatory and antitumor properties, was found to inhibit proteasome activity and induce the expression of hsp genes in the amphibian model system, Xenopus laevis. Treatment of Xenopus kidney epithelial A6 cells with WA produced an increase in the accumulation of ubiquitinated protein and a significant decrease in chymotrypsin-like activity. Furthermore, immunoblot analysis revealed that WA induced HSP30 and HSP70 accumulation. For example, cells treated with 5 μM WA for 18 h resulted in the optimal accumulation of HSP30 and HSP70. Northern blot analysis revealed that exposure of cells to 5 μM WA induced hsp30 and hsp70 mRNA accumulation in a time-dependent manner up to 12 h. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in WA-induced hsp gene expression in A6 cells, since pretreatment with the HSF1 inhibitor, KNK437, reduced the accumulation of HSP30 and HSP70. Also, WA acted synergistically with mild heat shock to enhance HSP accumulation to a greater extent than the sum of both stressors individually. In cells recovering from WA, the relative levels of HSP30 and HSP70 accumulation remained elevated from 6 to 12 h after removal of WA. Immuocytochemical analysis and laser scanning confocal microscopy revealed that WA-induced HSP30 accumulation occurred primarily in the cytoplasm with some staining in the nucleus in a granular or punctate pattern. Prolonged exposure to WA resulted in some disorganization of the actin cytoskeleton as well as large cytoplasmic HSP30 staining structures in some cells. Prior exposure of cells to WA treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 °C. In conclusion, this study has shown that WA can induce an inhibition of proteasome activity and an increase hsp gene expression. Activating the heat shock response is a potential avenue for novel drug therapies, which can confer cytoprotection in disease states involving cytotoxic protein aggregation.