Abstract
Various pathways can repair DNA alkylation by chemotherapeutic agents such as temozolomide (TMZ). The enzyme O6-methylguanine methyltransferase (MGMT) removes O6-methylated DNA adducts, leading to the failure of chemotherapy in resistant glioblastomas. Because of the anti-chemotherapeutic activities of MGMT previously described, estimating the levels of active MGMT in cancer cells can be a significant predictor of response to alkylating agents. Current methods to detect MGMT in cells are indirect, complicated, time-intensive, or utilize molecules that require complex and multistep chemistry synthesis. Our design simulates DNA repair by the transfer of a clickable propargyl group from O6-propargyl guanine to active MGMT and subsequent attachment of fluorescein-linked PEG linker via ”click chemistry.” Visualization of active MGMT levels reveals discrete active and inactive MGMT populations with biphasic kinetics for MGMT inactivation in response to TMZ-induced DNA damage.
Highlights
DNA alkylating agents such as temozolomide (TMZ) are essential first-line chemotherapeutic agents used to treat several cancers such as glioblastomas (GBM) and melanomas [1]
These cells were incubated with O6 -benzylguanine (O6 BG), an methylguanine methyltransferase (MGMT) inhibitor, for varying durations (0–165 min, see Figure 2), and O6 -propargyl guanine (O6 PGG) was added to the standard culture medium
We discovered that PFA-fixed, phosphorylated/inactive MGMT could be reactivated and become a substrate for O6PGG, by incubation with alkaline phosphatase (AP)
Summary
DNA alkylating agents such as temozolomide (TMZ) are essential first-line chemotherapeutic agents used to treat several cancers such as glioblastomas (GBM) and melanomas [1]. These drugs mediate their cytotoxicity by forming covalent DNA adducts. Diazomethane methylates the N3 -position of adenine and the N7 and O6 positions of guanine, produces up to 13 different DNA base adducts. Of these adducts, O6 -methylguanine (O6 MeG), accounts for
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