Abstract
Based on the high rate of chromosomal defects in MDS, inherent chromosomal instability (CIN) has been hypothesized as a key pathophysiologic factor of clonal evolution. Predisposition to DNA damage may be primarily due to acquired/inherited weakness in DNA repair machinery; such insufficiency may become manifest after a long latency following cumulative exposure to genotoxic agents. Subsequent changes in chromosomal structure and stepwise acquisition of neoplastic features could lead to leukemic progression. Previously, a number of allelic polymorphisms in DNA repair genes were observed. These variants may lead to altered expression of corresponding proteins. Conversely, if DNA damage is a primary defect, upregulation of specific DNA repair enzymes may be compensatory. Irrespective of the initial pathogenetic defect, we theorized that broad analysis of DNA repair machinery in MDS may point towards specific lesions that could be a subject of more targeted studies. Therefore, we examined levels of DNA repair enzymes using gene expression arrays. For proper comparisons, CD34 cells from 10 MDS patients (4 RA, 6 RAEB/RAEBt) and healthy controls were used. Expression array results were confirmed by Taqman PCR. Reference expression was established by pooling RNA from 12 controls. For more targeted analysis, A-CGH based genomic scan was used to better assess the extent of DNA damage in patients. The expression of 22 out of 113 DNA repair genes tested was detectable at levels >1,5X background; 2-level normalization of gene expression was performed according to variation of mRNA input (housekeeping gene-ACTB) and inter-assay variation in the signal intensity (biotinylated artificial sequence -BAS2C). Our combined standard sample was validated against individual controls; signals <1,5X pooled expression were obtained. Using expression levels of normal CD34 cells as a reference we found that 19 genes were upregulated in concordant fashion. The most dramatically increased genes included APEX, ATM, XRCC1, XRCC5 and MPG. This finding favors the theory that overexpression of the DNA repair machinery is a compensatory event to cope with a primarily increased level of DNA damage. When we subgrouped MDS patients according to FAB criteria, the expression of DNA repair genes (e.g., CIB1, ERCC1, SUMO1) increased with the malignant progression. For further analysis we have defined CIN phenotype by the presence of large or multiple small defects as determined by A-CGH. When patients with CIN vs. those with normal karyotype were compared, we found that chromosomal damage was not accompanied by a higher expression of DNA repair genes. MPG was most dramatically upregulated in all MDS patients. This gene involved in excision of methylated bases can induce single stranded breaks (SSB) and increase sensitivity to alkylating agents. Our finding suggest that either increased purine methylation induces a compensatory mechanism (MPG upregulation) or that overactivity of MPG itself results in increased base excision. Alternatively, overexpression of MPG may lead to SSB especially because downstream genes (e.g. XRCC3 or DNA ligase III) were not accordingly upregulated. In conclusion, our studies form a basis for further analysis of clinical phenotypes associated with upregulation of specific DNA repair genes and may indicate possible therapeutic targets in molecularly defined subtypes of MDS.
DNA damage shifts circadian clock time via Hausp-dependent Cry1 stabilization
