The effect of thiopurine drugs on DNA methylation in relation to TPMT expression.

The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2'-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5'-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.

Epigenetics as a mechanism driving polygenic clinical drug resistance.

Aberrant methylation of CpG islands located at or near gene promoters is associated with inactivation of gene expression during tumour development. It is increasingly recognised that such epimutations may occur at a much higher frequency than gene mutation and therefore have a greater impact on selection of subpopulations of cells during tumour progression or acquisition of resistance to anticancer drugs. Although laboratory-based models of acquired resistance to anticancer agents tend to focus on specific genes or biochemical pathways, such 'one gene:one outcome' models may be an oversimplification of acquired resistance to treatment of cancer patients. Instead, clinical drug resistance may be due to changes in expression of a large number of genes that have a cumulative impact on chemosensitivity. Aberrant CpG island methylation of multiple genes occurring in a nonrandom manner during tumour development and during the acquisition of drug resistance provides a mechanism whereby expression of multiple genes could be affected simultaneously resulting in polygenic clinical drug resistance. If simultaneous epigenetic regulation of multiple genes is indeed a major driving force behind acquired resistance of patients' tumour to anticancer agents, this has important implications for biomarker studies of clinical outcome following chemotherapy and for clinical approaches designed to circumvent or modulate drug resistance.

CpG-island methylation and epigenetic control of resistance to chemotherapy.

Aberrant methylation of CpG islands (CpG-rich regions of DNA associated with the promoters of many genes) is associated with transcriptional inactivation of genes involved in tumour development. Genes involved in key DNA damage response pathways, such as cell-cycle control, apoptosis signalling and DNA repair can frequently become epigenetically silenced and methylated in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, chemotherapy itself may exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Clinical trials of epigenetic therapies are now in progress, and epigenetic profiling using DNA methylation will provide guidance on optimization of the use of these therapies with conventional chemotherapy, as well as helping to identify patient populations who may particularly benefit from such approaches.

During U937 monocytic differentiation repression of the CD43 gene promoter is mediated by the single-stranded DNA binding protein Pur alpha.

Human CD43 is an abundant, heavily glycosylated molecule expressed exclusively on the surface of leucocytes. When leucocytes are at rest, CD43 acts to prevent intercellular interaction but during leucocyte differentiation such cell-cell interaction is facilitated by CD43. This change in the function of CD43 is mediated in part by a reduction in its level of expression. Previous studies have implicated proteolytic cleavage events at the cell surface in causing such reduction. Here, we report that, in an in vitro model of leucocyte differentiation, CD43 mRNA levels were also subject to reduction. Specifically, we demonstrated that within 48 h of the cell line U937 being induced to differentiate along the monocytic pathway, CD43 mRNA levels were reduced by 69%. This decline coincided with a decrease in the activity of the CD43 gene promoter mediated by the single-stranded DNA binding protein Pur alpha. Previously, we have demonstrated that Pur alpha mediates induction of the CD11c beta 2 integrin promoter during U937 differentiation. Consequently, Pur alpha represents a potential means by which the induction of pro-adhesive molecules and the repression of anti-adhesive molecules is co-ordinated during leucocyte differentiation.

CD43 gene expression is mediated by a nuclear factor which binds pyrimidine-rich single-stranded DNA.

CD43 is a leukocyte-specific surface molecule which plays an important role both in adhesion and signal transduction. We have identified a site spanning nucleotides +18 to +39 within the human CD43 gene promoter which in vitro is hypersensitive to cleavage by nuclease S1. Repeats of this region are sufficient to activate expression of a heterologous promoter in CD43-positive cell lines. Two nuclear factors, PyRo1 and PyRo2, interact with the hypersensitive site. PyRo1 is a single-stranded DNA-binding protein which binds the pyrimidine-rich sense strand. Mutation analysis demonstrates that the motif TCCCCT is critical for PyRo1 interaction. Replacement of this motif with the sequence CATATA abolishes PyRo1 binding and reduces expression of the CD43 promoter by 35% in Jurkat T lymphocytic cells and by 52% in the pre-erythroid/pre-megakaryocytic cell line K562. However, this same replacement failed to affect expression in U937 monocytic cells or in CEM T lymphocytic cells. PyRo1, therefore, exhibits cell-specific differences in its functional activity. Further analysis demonstrated that PyRo1 not only interacts with the CD43 gene promoter but also motifs present within the promoters of the CD11a, CD11b, CD11c and CD11d genes. These genes encode the alpha subunits of the beta2 integrin family of leukocyte adhesion receptors. Deletion of the PyRo1 binding site within the CD11c gene reduced promoter activity in T lymphocytic cells by 47%. However, consistent with our analysis of the CD43 gene, the effect of this same deletion within U937 monocytic cells was less severe. That PyRo1 binds preferentially to single-stranded DNA and sequences within the CD43 and CD11 gene promoters suggests that expression of these genes is influenced by DNA secondary structure.

Fragments from alpha-actinin insert into reconstituted lipid bilayers.

Recent experiments have indicated that alpha-actinin interacts with phospholipid membranes. Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281-300 and 720-739 of the primary amino acid sequence on smooth muscle alpha-actinin. Having expressed these regions as fusion proteins with schistosomal GST (glutathione S-transferase), we used differential scanning calorimetry (DSC) to investigate their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers. Calorimetric measurements showed that as fusion protein concentration increased, the main chain transition enthalpy decreased and chain melting temperatures shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membranes. Centrifugation assay and subsequent SDS/Page chromatography confirmed this finding.

Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers.

Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity.