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This dissertation, Molecular Study of Pi-class Glutathione-S-transferase in Endometrial Carcinoma by Kwan-yi, Queeny, Chan, 陳君怡, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Molecular study of pi-class glutathione-S-transferase in endometrial carcinoma submitted by Chan Kwan Yi, Queeny for the Degree of Master of Philosophy at the University of Hong Kong in August 2003 pi-class glutathione-S-transferase (GSTP1), located on chromosome 11q13, is coding for a phaseII metabolic enzyme that detoxifies reactive electrophilic intermediates. The protein also interacts with steroid hormones thus minimizing the effects of short-term fluctuations of hormone levels in human body. The role of GSTP1 has not been studied in endometrial carcinoma (EmCA), a hormone related cancer. In the first part of this project, we aimed at evaluating the effect of hypermethylation on different subtypes of endometrial carcinoma, by determining the level of GSTP1 protein expression by immunohistochemistry (IHC) on tissue microarray, the level of its mRNA expression by real-time reverse transcription polymerase chain reaction (Q-RT-PCR), its methylation status by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing, 2 and existence of somatic mutations by cDNA sequencing. Fresh frozen and paraffin-embedded tissues of 125 cases of EmCA including 90 cases of purely endometrioid type, 20 cases of endometrioid carcinoma with focal squamoid, mucinous or secretory differentiation, and 15 cases of non-endometrioid adenocarcinoma of serous or clear cell carcinomas were retrieved. Our results showed that 69.6% (87/125) of the cases showed reduced protein expression, 64% (16/25) showed reduced mRNA expression, and 30.9% (30/97) of cases were hypermethylated as demonstrated by at least one of the six MS-PCR reactions. Hypermethylation found in tumor cases was statistically correlated with reduced protein expression (p=0.008) and reduced mRNA expression (p=0.029). There may be additional mechanisms accounting for the reduced GSTP1 expression in EmCA. Our cDNA sequencing, however, did not reveal mutations in coding region of the gene. Methylation status and expression level were correlated with the extent of myometrial invasion, but not with other clinicopathological data of patients. To conclude, reduced expression of GSTP1 was a relatively common phenomenon in EmCA. Mechanisms other than methylation may account for the reduced expression. In the second part of our study, we aimed at determining the susceptibility of developing endometrial carcinoma in association with a particular GSTP1 allele. Recent studies have identified a single nucleotide polymorphism of the gene that 3 results in an amino acid substitution from isoleucine to valine at codon 105 (I105V), and demonstrated that such valine substitution may ultimately lead to substrate-specific functional change in the activity of the enzyme and increasing the risk for developing cancers. One hundred and thirty-nine age-matched pairs of formalin-fixed, paraffin-embedded tissue blocks were obtained from normal controls and patients with histologically confirmed endometrial cancers. Genotypes were determined using a polymerase chain reaction/ restriction fragment length polymorphism (PCR-RFLP) assay. The relative risk (estimated by odd ratios) of developing endometrial cancers for the GSTP1 genotype was calculated using logistic regression models. Experimental data showed that allelic frequencies were different in normal controls (Ile-0.802, Val-0.198) and cance

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