UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (similar to 22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.