A candidate gene based investigation of aberrant DNA methylation in the pathogenesis of primary cutaneous T-cell lymphoma
Primary cutaneous T-cell lymphoma (CTCL) is a clinically heterogeneous malignancy of mature skin-homing T-cells. Mycosis fungoides (MF) is an indo¬lent subtype of CTCL whilst Sezary syndrome (SS) is an aggressive leukaemic variant characterised by the presence of malignant Sezary cells in the periph¬eral blood. A role for epigenetic mechanisms in the pathogenesis of CTCL has been proposed but not extensively investigated. In this thesis, the contribution of DNA methylation to the regulation of SHP-1, Fas and PLS3, which have been implicated in the pathogenesis of CTCL, was examined. Gene expression was assessed on the mRNA and protein levels using qPCR and flow cytometry re¬spectively whilst DNA methylation was evaluated using bisulphite conversion and Pyrosequencing. Contrary to results in CTCL cell lines, primary malignant cells from CTCL patients did not display aberrant DNA methylation or dysregu-lated expression of SHP-1, a negative regulator of STATS signalling. Dysregula-tion of Fas, a key regulator of apoptosis, was observed in 34/47 SS patients with 13/47 showing over-expression compared to healthy controls and 21/47 showing under-expression, attributed to the positional hypermethylation of five CpG din-ucleotides in the Fas CpG island. PLS3, an actin-binding protein not normally expressed in any haematopoietic cell, was aberrantly expressed in malignant cells from 21/35 SS patients and 3/8 MF patients with demonstrated clonal blood in¬volvement. CpG dinucleotides 95-99 in the PLS3 CpG island were differentially methylated between healthy lymphocytes and keratinocytes, and hypomethyla-tion of these CpG dinucleotides was observed in SS patients expressing PLS3. To investigate expression of PLS3 on the protein level a novel anti-PLS3 anti¬body was raised and optimised for use in Western blotting, flow cytometry and immunofluorescence. In conclusion, these data demonstrate that SHP-1 is not regulated by methylation in primary CTCL cells whilst Fas is dysregulated by hypermethylation of five key CpG dinucleotides and PLS3 is dysregulated by hy-pomethylation of CpG dinucleotides 95-99.
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