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Neuroblastoma (NB) is a childhood cancer with a highly complex nature. High-risk NB patients undergo intensive treatment regimens that are often followed by long-term side effects. This, in addition to the emergence of resistant cancer cells, highlights a need for novel therapeutic targets and treatment strategies to improve outcome in NB. P21 is a cyclin-dependent kinase inhibitor considered to play a role in tumor resistance and aggressiveness due to its involvement in cell cycle and/or apoptosis. This project aimed to explore the expression of endogenous p21 in high-risk NB cell lines and whether p21 could be a therapeutic target for high-risk NB. Endogenous p21 levels were investigated using RT-qPCR and quantitative immunocytochemistry in eight high-risk NB cell lines. A small molecular inhibitor of p21, UC2288, was used in these cell lines to investigate tumour cell viability following p21 inhibition. In addition, combination treatment with UC2288 and the chemotherapy drug cisplatin was performed on resistant NB cell lines. Our results show variable expression of p21, where cell lines with high endogenous p21 expression showed sensitivity to single agent treatment with cisplatin or UC2288. Moreover, resistant NB cell lines showed lower endogenous p21 expression, however, combination treatment with UC2288 and cisplatin showed reduced viability, indicating sensitivity to combination treatment. This project highlights the potential of using p21 as a therapeutic target as well as a predictive biomarker in high-risk NB.

<p>This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca²⁺pumps (PMCA) needed to understand the Ca²⁺signalling in coronary artery.</p> <p>PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.</p> <p>In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.</p> <p>Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca²⁺-Mg²⁺-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca²⁺ concentration in endothelial cells.</p> <p>Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)