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In vitro functional analysis of TP53 transfected human cancer cells

Among the genetic mutations involved in carcinogenesis, TP53 mutation is a frequent event in many types of cancer. P53 is a transcription factor that regulates activities such as cell cycle arrest, apoptosis, DNA repair and angiogenesis. The majority of TP53 mutations are missense mutations that accumulate in cancer and are often retained in distant metastases. The effects of the mutant p53 proteins include loss of function, dominant-negative effects over wild-type (WT) p53 and possible acquisition of new properties (gain-of-function). However, some of these properties may differ from one mutant p53 protein to another. These differences could have implications for the in vivo behaviour of tumours carrying particular mutations and hence patient prognosis. The aim of this project was to investigate the phenotypic variation between cells transformed with different p53 mutants. This was achieved by constructing a range of TP53 mutants (R175H, G245S, R248W, R248Q, R273H, R282W) using PCR-based mega-primer site directed mutagenesis. These mutants were cloned into a mammalian bi-cistronic expression vector (designed for the co-expression of WT and mutant TP53 from a single plasmid) to allow transient expression in NCI-H358 cells (p53 null). Regard to the method for PCR site directed mutagenesis, the main technical difficulty with conventional methods was the insufficiency of the mutant TP53 product yield (75%). This thesis has modified these methods by carrying over the start template to a second round of PCR and increasing the MgCl2 concentration. This modified PCR-based site directed mutagenesis method has demonstrated an increased mutant TP53 product yield (100%). The tetracycline expression system is the most widely used for conditional inducible systems in mammalian cells, although high background expression has been a main problem. The ecdysone inducible system potentially allows for the study of the conditional expression of the exogenous reporter gene even though it may be cell lethal or alter the phenotype during the selection of transfectants. This system relies on two independent transfections of two plasmids namely pVgRXR and pIND. However, disruption of the regulatory element within the plasmid during stable integration can result in silence or high background expression of the exogenous reporter gene. A previous study reported a transient luciferase reporter assay to screen the cell line stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. This thesis has demonstrated a real time RT-PCR strategy to screen for the background expression problem associated with the ecdysone expression system. However, due to the project’s time limitations, a transient expression system rather than a stable expression system was used. The metastasis related cellular activity of WT/mutant TP53 transfected NCI-H358 cells was examined using a range of in vitro functional assays including a proliferation assay, a p21 promoter binding activity assay, a colony formation assay, and a migration assay. To extend the study, this thesis also employed real-time RT-PCR to examine the mRNA expression level of three metastatic related genes, VEGF, HER-2, and E-cadherin, in the WT/mutant TP53 transfected NCI-H358 cells. The results showed that different WT/mutant TP53 transfected cell linse could contribute to markedly different cellular activity. Among these mutants, R175H produced the highest cellular proliferation activity, the strongest dominant-negative activity over the WT on the p21 promoter binding activity and apoptosis activity, and the greatest effect on cellular migration. Furthermore, the real-time PCR results showed that the WT p53 inhibited transcription of key metastasis-related genes such as VEGF and HER-2. Considered with recent literature, this led me to postulate a feedback amplification cycle involving defective p53 and HER-2 mRNA expression. In conclusion, cancer cells with the R175H mutant could contribute to aggressive tumours. This conclusion, based on the in vitro data, is consistent with some clinical observations and animal model experiments. In the past few years it has become apparent that epigenetic changes also play a vitally important role in the cancer developmental process. Recent studies have reported the p53 protein can contribute in methylation which is one of the processes involved in epigenetic modification. This thesis employed a very new PCR-based AMP technique to examine the change of the global genome methylation pattern as a result of knocked-out p53 protein. The results showed defective p53 protein expression may associate with the global genome methylation pattern changes. However, it is important to note that antibiotic reagents, which were used for stable transfectant selection, could also contribute to the global genome methylation changes. In conclusion, this thesis has successfully developed two new methods. One allows the generation of a genetic mutant construct using PCR-based site directed mutagenesis while the other screens the tightly regulated ecdysone reporter system. In terms of effect of p53 in in vitro cell activity, this thesis has postulated that the R175H mutation is associated with much more aggressive metastatic cellular activity. Finally, this thesis also reported that loss of p53 expression could also result in changes in the global genome methylation pattern.

Identiferoai:union.ndltd.org:ADTP/279190
CreatorsRichard Lai
Source SetsAustraliasian Digital Theses Program
Detected LanguageEnglish

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