The main goal of this project is to investigate whether the cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) genes are risk factors for pathobiology of endometrial cancer. The rationale is that CYP1A1 and CYP1B1 catalyze the hydroxylation of estrogen to highly carcinogenic 2-hydroxy catechol estrogen (2-OH-E2) and 4-hydroxy catechol estrogen (4-OH-E2) metabolites, respectively. However, such studies are lacking in endometrial cancer. Based on our preliminary data and prior publications, we hypothesize that CYP1A1 and CYP1B1 genes are risk factor for pathobiology of endometrial cancer.
Specific Aim # 1. To investigate whether the CYP1A1 and CYP1B1 genes are hyper-activated in endometrial cancer. We hypothesize that the CYP1A1 and CYP1B1 genes are hyper-activated during malignant transformation of the endometrium. To test this hypothesis, we will: (a) Analyze the level of CYP1A1 and CYP1B1 mRNA expression by real-time PCR (for quantification) in benign endometrium and in different stages and grades of endometrial cancer. In addition, in situ hybridization will be used to localize CYP1A1 and CYP1B1 mRNA expression in endometrial tissues, (b) Analysis of protein expression of CYP1A1 and CYP1B1 in benign endometrium and in different stages and grades of endometrial cancer using immunohistochemistry (for localization) and western blotting for quantification. (c) Analysis of the level of CYP1A1 and CYP1B1 enzymatic activity in endometrial tissues by biochemical techniques as well as determine the level of estrogen metabolites (2-OH-E2 and 4-OH-E2) in endometrial tissues by gas chromatography methods, (d) To analyze whether CYP1A1 and CYP1B1 genes, protein, enzymatic activity and/or the levels of 2-OH-E2 and 4-OH-E2 metabolites in endometrial cancer are predictors of recurrence and patient survival.
Specific Aim # 2: To investigate whether single nucleotide polymorphisms of the CYP1 Al and CYP1B1 genes are risk factors for endometrial cancer. We hypothesize that the analysis of SNPs in the CYP1A1 and CYP1B1 genes can identify populations with a higher risk of endometrial cancer. To test this hypothesis, we will: (a) Analyze SNPs of the CYP1A1 and CYP1B1 genes in blood and tissue DNA from benign and endometrial cancer patients using PCR-RFLP (restriction fragment length polymorphism) and direct genomic sequencing, (b) Analyze whether SNPs of CYP1A1 and CYP1B1 genes are associated with altered enzymatic activity and the altered production of estrogen metabolites in endometrial cancer, (c) Analyze whether SNPs of the CYP1A1 and CYP1B1 genes are associated with endometrial cancer recurrence and patient survival.
Specific Aim # 3: To investigate whether polymorphic variants of the CYP1A1 and CYP1B1 genes can modulate estrogen hydroxylase activity. We hypothesize that SNPs in exon regions of CYP1A1 and CYP1B1 genes can modulate their enzymatic activity in endometrial cancer. To test this hypothesis we will express wild-type and polymorphic variant forms of CYP1A1 and CYP1B1 (which contain a NH2-terminal hexahistidine tag) using a ThioFusion expression vector (pThioHis). CYP1A1 and CYP1B1 mRNA isolated from normal endometrium will serve as the source of wild-type cDNAs, which will be inserted into the pThioHis expression vector. We will then use the wild-type pThioHis CYP1A1 and CYP1B1 expression plasmids as templates to generate polymorphic variants of CYP1A1 and CYP1B1 using site-directed mutagenesis and determine their estrogen hydroxylase activity. This project will provide us with novel mechanisms involving the pathobiology of endometrial cancer.
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