Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Title page, table of contents and introduction only. The complete thesis in print form is available from the University of Adelaide Library. / Apoptosis and inflammation have been considered to be linked mechanisms. Defective apoptosis may result in cell accumulation and prolonged half life of inflammatory cells in rheumatoid arthritis (RA). A similar phenomenon is seen in malignancy. Therefore, defects in apoptosis pathways that have been proposed to contribute to the pathogenesis of malignancy may also be seen in RA. It has been reported by many studies that treatments that induce apoptosis of malignant cells are advantageous for cancer treatment and this suggests that targeting apoptosis pathways may be important in the management of a variety of pathologies that involve abnormalities in cell proliferation. While apoptosis induction has been a common mechanism in the treatment of cancer, it has only recently been seriously considered to be effective in regulating proliferative cells in inflammation. This thesis provides important information regarding the TRAIL mediated pathway of apoptosis and possible factors that modulate the ability of this pathway to induce apoptosis in RA. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1286764 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2007

The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (MScMed)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--Tufts University, 1999. / Adviser: Shuk-Mei Ho. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 169-171). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR19833

Erlangen, Nürnberg, Universiẗat, Diss., 2006. / Enth. 1 Sonderabdr. aus: Strahlentherapie und Onkologie ; 181. 2005. - Beitr. teilw. dt., teilw. engl.

Erlangen, Nürnberg, University, Diss., 2006.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.