Differential hMLH1 Gene Expression after Temozolomide Selection Linked to Microsatellite Instability in a Subset of Hematopoietic Stem/Progenitor Cells in Old Versus Young and Cancer Versus Normal Patient Samples.

The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34⁺ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O⁶-Benzylguanine (BG) to remove O⁶-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O⁶-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O⁶-methylguanine (O⁶-mG) lesions that persist in the presence of BG. These O⁶-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.