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Title: A multiplex comparative proteomic analysis of hypoxia influence in the presence and absence of p53 in HCT116 Cells
Authors: TAN WEE WEE
Keywords: colorectal cancer, Hypoxia, iTRAQ, p53, HCT116
Issue Date: 10-Jun-2008
Source: TAN WEE WEE (2008-06-10). A multiplex comparative proteomic analysis of hypoxia influence in the presence and absence of p53 in HCT116 Cells. ScholarBank@NUS Repository.
Abstract: Cells are constantly maintained and renewed in our body under a stringent homeostatic regulation. In the event when cellular damages are beyond repairs, these cells will be destroyed via the programmed cell death (PCD) pathway. In cancer, the PCD pathway becomes dysfunctional due to genetic mutations. Consequently, cells proliferate uncontrollably and lead to disruption of the vascular network. This results in the formation of hypoxic microenvironments within the tumor due to insufficient oxygen supply to the cells and the presence of hypoxic regions has been shown to correlate with poor prognosis and therapeutic resistance. Cellular activities of cancer cells undergo changes to cope with the oxygen-deprived (hypoxia) condition and these changes are achieved mainly by the action of hypoxia-inducible factor-1 (HIF-1), a transcription factor. In the presence of hypoxia, apoptotic-resistant tumor cells are selected, such as through the attenuation of p53 apoptotic response. However, attempts to confirm the relationship between p53 and hypoxia/HIF-1 have met with conflicting results. In this study, we investigate the differential gene expression in cultured human colorectal cancer cells, HCT116, subjected to hypoxic condition using isobaric tags (iTRAQ) and mass spectrometry. Using p53 knockout (KO) cells, we also examine the elusive relationship between hypoxia and p53 by analyzing their protein profiles. At 95% C.I., a total of 217 proteins were identified in our iTRAQ experiments and of which, the expression levels of 54 proteins were found significantly altered with at least 30% fold change in terms of protein abundance. Among the significantly affected proteins, 14 were potentially regulated by hypoxia and this includes the known hypoxia affected proteins, PGK1, LDHA, and FAS. Fifteen proteins were found potentially regulated by p53 and the remaining 25 proteins were affected by both hypoxia treatment and the presence of p53. An ontology analysis of these 54 proteins revealed that they were mainly involved in the regulation of cellular growth and proliferation. Downstream validation analysis using RT-PCR and immunoblotting assays further confirmed the observations in our iTRAQ results. Both RT-PCR and immunoblotting results strongly indicate that ANXA2 and PCBD1 may be novel interacting targets of p53 while the regulation of EFHD2 and CKS2 may be influenced by hypoxia (1% O2) treatment. Therefore, we proposed that these distinct differentially expressed proteins may be used as potential biomarkers and/or therapeutic targets in colorectal cancer.
Appears in Collections:Master's Theses (Open)

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