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Cytochrome P450 mRNA profile in human breast cancer cell lines.

Warasiha, Benjamart

Authors

Benjamart Warasiha



Contributors

Morag C.E. McFadyen
Supervisor

Rachel M. Knott
Supervisor

Abstract

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various compounds, both endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen). Intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aims of this thesis were to firstly profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs), and secondly to compare this profile with normal breast cells. This was achieved using a qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed that CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA were present in all cell lines. The study also showed that CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in certain BCCs that were characterised by oestrogen receptor (ER) - ER-positive Caucasian and ER-negative African-Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities between the ER-positive Caucasian and ER-negative African-Caribbean BCCs, which warrant further investigation. The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 BCCs were used in order to determine the appropriate nuclear receptors - i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha. The findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. Additionally, pre-treatment of these two cell lines - using the RNA synthesis inhibitor actinomycin D, followed by the agonists - showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels. It also inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast to this, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these same agonists. These results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type-specific regulatory mechanisms, and there is evidence for differential regulation of the splice variants.

Thesis Type Thesis
Institution Citation WARASIHA, B. 2008. Cytochrome P450 mRNA profile in human breast cancer cell lines. Robert Gordon University, PhD thesis.
Keywords Cytochrome P450 enzymes; Breast cancer treatments; Cell regulatory mechanisms

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Copyright Statement
Copyright: the author and Robert Gordon University





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