Abstract
hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase–PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1–MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.
Highlights
Genomic DNA is under constant attack by reactive oxygen species that occur naturally as by-products of aerobic metabolism
Increased production of reactive oxygen species is widely considered to be a consequence of malignant transformation in many cancer types,1,2 and efficient repair of oxidized DNA lesions is considered critical for cancer cell survival
The 8-oxodG:C pairs are thought to be mainly detected and further processed by 8-oxoguanine DNA glycosylase (OGG1) that performs the first step of base excision repair (BER), removing the oxidized guanine paired with cytosine
Summary
Genomic DNA is under constant attack by reactive oxygen species that occur naturally as by-products of aerobic metabolism. No considerable changes were observed in the expression of other DNA glycosylases (Figure 5a) These results hMYH and hMTH1 functionally collaborate to sustain cell survival in a T-ALL cell line implicate NEIL1 in the cellular response to excess genomic oxidized lesions, caused here by MTH1 and MYH double KD. We observed concurrent KD of both proteins induced more cell death compared with individual depletions in both hairpin sets (Figure 2) These observations support the hypothesis that hMYH function is required for cell survival when hMTH1 activity is suppressed. We speculate that cells, with potential increased 8-oxodG levels caused by hMTH1 or hMYH loss, may suppress NEIL1 expression to avoid incised hydantoin lesions (converted to DNA single-strand breaks) to cause replication fork collapse and DNA double-strand breaks. This study provides evidence for the functional collaboration between hMYH and hMTH1 to maintain survival in cancer cells with MMR defective backgrounds, in particular T-ALL Jurkat A3 cells
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