Hypermethylation of DNA and Increased Expression of DNMT1 May Represent a Protective Mechanism from Oxidative Stress and DNA Damage in MDS.

Epigenetic changes including altered cytosine methylation have been hypothesized to play a pathogenic role in myelodysplastic syndrome (MDS) and other myeloid malignancies. While the specific functional consequences of CpG island methylation depend on the pattern of silenced promoters, the triggers for changes in the methylation program are unknown. DNA damage is likely to interfere with faithful heritability of cytosine methylation patterns during cell division. For example, formation of 8-oxoguanine (8-OG) in CpG islands may result in hypomethylation through inhibition of methylation-sensitive human maintenance methyltransferase DNMT1. Alternatively, hypermethylation may be a part of cellular response to oxidative damage e.g., through stimulation of DNMT1 activity. When we investigated expression of various DNA repair genes in highly purified CD34+ progenitor and stem cells of patients with MDS (19 RARS/RA, 16RAEB1/2/sAML, 2 CMML) using expression arrays, we found a significant and concordant up-regulation of N-methylpurine DNA glycosylase MPG; this finding was confirmed by TaqMan PCR in 21/37 MDS patients tested. Over-expression of MPG may be in response to increased DNA alkylation, but MPG has been described to co-localize in methylated CpG islands and could reflect their increased methylation. We have stipulated that up-regulation of MPG is a consequence of ongoing DNA damage and chose to study other parameters that could support this theory. For example, incorporation of 8-oxoguanine can be measured by flow cytometry and when we used this method, we determined that an increased oxidative DNA load/stress was found in 67% of all MDS cases studied (n=24) and correlated with advanced stage of MDS (p<.006), cytopenia (e.g., neutropenia p<.002) and a greater propensity for AML transformation (p<.02). We also studied expression of DNA glycosylase/AP lyase OGG1 with the capacity to excise oxidized guanine from DNA and found up-regulation of OGG1 mRNA levels in 11/37 patients. To investigate whether oxidative/alkylating DNA damage is indeed associated with alteration of methylation we have measured 2 parameters:

• global methylation using an ELISA-based colorimetric assay specific for methylcytosine and

• expression of DNMT1 using TaqMan PCR.

We determined that patients with over-expression of OGG1 were characterized by lower expression of DNMT1 (>2x control levels) and inversely correlated with global methylation levels. In contrast, those patients with over-expression of DNMT1, a corresponding increase in global methylation was observed. In sum, our results are consistent with the inverse relationship between hypermethylation and oxidative stress or alternatively that hypermethylation may be a protective response to an ongoing DNA damage in a subset of MDS patients.