Abstract
Abstract
Increased frequency of chromosomal lesions in MDS points towards defects in the DNA repair machinery as pathogenetic factors responsible for clonal evolution. Base excision repair (BER) is a possible mechanism of genomic instability. Upon genotoxic stress or due to defective function of the components of the BER, single-stranded breaks accumulate and are converted into double stranded breaks during mitosis. Oxidative damage may result in generation of 8-oxoguanine (OG), the most abundant DNA lesion. OG can be quantitated in hematopoietic cells using flow cytometry and we found significantly increased content of OG in patients with MDS (n=29, p=.0088). We stipulated that this observation may be a result of overwhelmed repair mechanism due to its insufficiency or inability to handle significantly increased oxidative stress. Here, we investigated this phenomenon in a subgroup of patients with increased genomic OG content (i.e. OG fluorescence >mean+2SD of controls). Remarkably, these patients were characterized by advanced stage of MDS (p=.0027), more profound cytopenias (neutropenia p=.001, thrombocytopenia p=.014) and propensity for AML transformation (p=.036). As OGG1 is the initial glycosylase that excises OG, we investigated the expression levels of this enzyme in highly purified CD34+ progenitor and stem cells in controls and patients with a high OG score; we found two expression patterns allowing for further sub-classification of affected patients. Low OGG1 (24/34) pointed towards a subset of patients with an inherent lesion that required further investigation. In contrast high OGG1 levels, found in 10/34 patients, were either consistent with an appropriate response to ongoing oxidative stress or the effect of a positive feedback due to low enzymatic activity. The SNP S326C of OGG1 has been associated with impaired function of the enzyme and compensatory upregulation of OGG1 transcription. This SNP is present in heterozygous form in controls at the frequency of 29%, but we found it in 8/10 patients with a high OGG1 expression, while 9/12 patients with wild-type had decreased expression (p=.01). In general, MDS patients (n=63) showed 6% homozygosity (vs. 0% in controls, p=.15) and 41% heterozygosity (vs 29% in controls, p=.24). Consequently, increased OG content could indicate a dysfunction of OGG1 resulting from the S326C SNP that cannot be adequately compensated. In fact, in 3/5 patients with OGG1 variant, an increased OG content and expression of OGG1 were found. Regardless of the activity of the OGG1, its upregulation could have various consequences to the downstream elements of the BER such as Polβ, which fills in the gaps left by the activity of endonucleases. Using Taqman RT-PCR performed on CD34+ cells; we tested Polβ expression in patients with upregulation of OGG1 and identified 2 groups of patients. The high Polβ group points toward overwhelming oxidative stress despite of adequate feedback response. In contrast, the low Polβ group could represent a subset of patients with defective upstream elements of the BER including endonuclease. This theory can be supported by elevated numbers of apurinic sites, detected by Elisa (0.54 +/− 0.70 in controls vs 1.5 +/− 2.1 in MDS AP sites/105bp). Taken together our results indicate that various lesions can contribute to the dysfunction of BER that occurs in MDS in the context of increased oxidative stress.
