Epigenetics and Expression of SFRP1 and PPARG and Epigenetic Effects of Glucocorticoids in B-cell Acute Lymphoblastic Leukaemia

Abstract

B-cell Acute Lymphoblastic Leukaemia (B-ALL), a cancer of immature B-lymphocytes, is the most common cancer in childhood. This cancer is characterised by widespread abnormalities of DNA methylation, when compared with non-cancerous blood cells.

DNA methylation is a chemical modification of the cytosine residues of DNA, and only cytosine residues immediately followed by guanine residues (so called CpG sequences or sites) undergo methylation. Methylation of CpG sites in gene promoter regions leads to non-expression of the methylated gene. DNA methylation abnormalities in cancers (such as B-ALL) have received significant attention over recent years, and have been shown to have significant biological effects in tumour cells, due to abnormal expression of the aberrantly methylated genes.

This project aimed to show that the putative tumour suppressor genes, SFRP1 and PPARG, showed increased DNA methylation in B-ALL cells, when compared with normal blood cells and that this was associated with reduced expression of these genes in B-ALL. The methylation of the gene promoters was determined by bisulphite sequencing and gene expression by qRT-PCR.

The results showed that PPARG and SFRP1 both show increased methylation in the gene promoter regions of B-ALL cells, when compared with normal blood cells. SFRP1 has previously been shown to show reduced expression in B-ALL and the qRT-PCR results showed that the PPARγ-1 transcript from the PPARG gene showed reduced expression in B-ALL cells, when compared with B-cells from normal blood as well as normal whole blood. Overall, it was concluded, on the basis of these results and others’, that SFRP1 and PPARG show reduced expression compared with normal blood cells, due to promoter methylation in B-ALL.

It has also been suggested in the literature that glucocorticoid drugs (analogues to the steroid hormone cortisol) can alter the methylation of CpG sites in individual genes (in non B-ALL cells). This is of interest in the context of B-ALL, as glucocorticoids are well known to be strong anti-leukaemia agents and are used in B-ALL treatment. Glucocorticoids are also known to affect the expression of many genes, an effect that is compatible with changing the DNA methylation of cells. Therefore, this project also aimed to show that the glucocorticoid dexamethasone could induce changes in DNA methylation in many genes within the genomes of B-ALL cells. Multi-gene methylation was measured using the, relatively new, RRBS technique with the NALM-6 human B-ALL cell-line with or without exposure to dexamethasone acting as my model of B-ALL. The results showed a number of methylation changes throughout the genome, with some particularly strong methylation changes observed in the promoter regions of the genes SPINT2, GATA3, IRX5, SOX13, GATM, PDGFA and DOCK10; genes implicated in cancer or in steroid-sensitive metabolism (such as energy metabolism). These results suggest that steroids do indeed alter the DNA methylation of B-ALL cells, which, if these results are replicated, is a novel mode of action of glucocorticoids in B-ALL treatment.