5-Aza-2′-deoxycytidine-induced genome rearrangements are mediated by DNMT1

Abstract

Observations that genome-wide DNA hypomethylation induces genomic instability and tumors in animals caution against the indiscriminate use of demethylating agents, such as 5-aza-2′-deoxycytidine (5-Aza-dC). Using primary mouse embryonic fibroblasts harboring a lacZ mutational reporter construct that allows the quantification and characterization of a wide range of mutational events, we found that in addition to demethylation, treatment with 5-Aza-dC induces γ-H2AX expression, a marker for DNA breaks, and both point mutations and genome rearrangements. To gain insight into the source of these mutations we first tested the hypothesis that the mutagenic effect of 5-Aza-dC may be directly mediated through the DNA methyltransferase 1 (DNMT1) covalently trapped in 5-Aza-dC-substituted DNA. Knock-down of DNMT1 resulted in increased resistance to the cytostatic effects of 5-Aza-dC, delayed onset of γ-H2AX expression and a significant reduction in the frequency of genome rearrangements. There was no effect on the 5-Aza-dC-induced point mutations. An alternative mechanism for 5-Aza-dC-induced demethylation and genome rearrangements via activation-induced cytidine deaminase (AID) followed by base excision repair (BER) was found not to be involved. That is, 5-Aza-dC treatment did not significantly induce AID expression and inhibition of BER did not reduce the frequency of genome rearrangements. Thus, our results indicate that the formation of DNMT1 adducts is the prevalent mechanism of 5-Aza-dC-induced genome rearrangements, although hypomethylation per se may still contribute. Since the therapeutic effects of 5-Aza-dC greatly depend on the presence of DNMT1, the expression level of DNA methyltransferases in tumors may serve as a prognostic factor for the efficacy of 5-Aza-dC treatment.

RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated Dnmt1 degradation and promoting apoptosis

“…Figure 2B shows that LCA exerted a significant dose-dependent inhibition of Ras-induced colony formation in RGS6 −/− MEFs while slightly reducing colony formation in WT MEFs at the highest dose used. Similar results were obtained with the non-selective Dnmt1 inhibitor 5-Aza-2’-deoxycytidine (5-Aza-dC)(30-32), though 5-Aza-dC did not affect colony formation in WT MEFs at doses sufficient to significantly impair transformation of RGS6 −/− cells (Fig. S3A).…”

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

“…Most of the DNA damage arises from the DNA-DNMT crosslinks which can block DNA synthesis and induce directly or indirectly DNA double-strand breaks, eventually leading to cell death (8). Decitabine has also been reported to enhance mutagenesis, mainly point mutations and genome rearrangements most likely due to protein-DNA crosslinks (9,10). The biochemical pathway that initiates repair of DNA-DNMT adducts has not been yet described in detail, however, it may involve DNA double-strand break repair factors at its late stages.…”

DNA Damage in Cancer Therapeutics: A Boon or a Curse?

“…Chemotherapeutic drugs such as alkylating agents (e.g., cyclophosphamide, cisplatin, and busulfan), DNA topoisomerase inhibitors (e.g., etoposide and mitoxantrone), and anthracyclines, which act by inducing DNA damage in cells, are all associated with a higher incidence of myeloid neoplasms. Interestingly, these drugs, as well as azacitidine, a DNA methylation inhibitor commonly used to treat MDS patients, can induce genetic rearrangements (29,30). Strikingly, these chemotherapy-induced myeloproliferative disorders are generally more resistant and have a worse prognosis than other cancers (29).…”