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  • br Phenotypic responses alkylation induced cell death and mu

    2021-06-10


    Phenotypic responses: alkylation-induced cell death and mutagenesis E. coli alkB mutants were isolated in a screen for strains specifically sensitive to the cytotoxicity of MMS but not UV-irradiation. This was the first indication that the AlkB protein is a primary cellular defence against alkylating agents [41], [79]. Indeed, alkB mutants of both E. coli and Caulobacter crescentus are profoundly sensitive to cell killing by MMS [41], [80]. Overexpression of AlkB in HeLa cells conveys striking protection against the cytotoxicity of MMS and DMS, but not against induction of sister chromatid exchanges [81]. Human ABH3 overexpressed in COS cells LED209 synthesis also conveyed a small increase in MMS resistance [82]. These observations indicate that removal of 1meA/3meC from DNA in both bacteria and mammalian cells prevents cytotoxicity. A controversial question is whether any of this protection results from repair of damaged RNA [62], [69]. Although profoundly sensitive to MMS-toxicity, E. coli alkB mutants are only weakly susceptible to MMS-induced mutagenesis [41], [42] resulting in small increases in GC to AT, GC to TA and AT to TA LED209 synthesis substitutions [42]. For comparison, an Ada Ogt mutant that lacks O6meG-DNA methyltransferase activity is 100-fold more susceptible to MMS-induced mutations than an AlkB mutant [42] (Fig. 5) despite the fact that MMS generates about 10-fold less O6meG than 1meA or 3meC in DNA [83]. So, in E. coli, 1meA and 3meC in DNA appear to be approximately 103-fold less likely to mispair in vivo than O6meG. A recent report seems to imply that AlkB has a greater impact on MMS-induced mutagenesis especially in SOS-induced cells although observed mutation frequencies [84] were similar to those reported earlier [41], [42]. Since the GC to AT and AT to TA mutations induced by MMS in the alkB mutant were dependent on umuC (PolV) expression, they may have resulted from by-pass of lesions by Pol V [84]. Due to the susceptibility of 3meC to deamination, and of 1meA to undergo the Dimroth rearrangement to N6-methyladenine at high pH, new methods to prepare and deprotect phosphoramidites of these modified bases have been developed [85]. This has allowed single alkylated bases, 1meA, 1etA, 3meC, 1meG or 3meT, to be inserted into a single-stranded DNA vector. Using these vectors, the modified bases were shown to be strong blocks to DNA replication in vivo[55] confirming several early observations in vitro[86], [87]. Low levels of DNA polymerase by-pass of 3meC and 1meG indicated that these lesions may be the source of the previously observed MMS-induced GC to AT and GC to TA mutations. Since 1meA was rarely by-passed, AT to TA transversions were ascribed to 3meT [55], however 3meT along with 1meG are very rare MMS-induced lesions [83]. The 3meC lesion appeared to be more mutagenic than 1meA in DNA [55]. Since 3meC is readily deaminated to 3-methyluracil it is unclear whether deamination of 3meC during the extended experimental procedures employed accounts for the GC to AT transition mutations. Alkylating agents are used in the treatment of several human cancers, and attempts have been made to enhance these therapies by inhibiting repair of the toxic alkylated DNA lesions. Such studies have not been performed with AAG or ABH2/ABH3 but inhibitors of the human O6meG-DNA alkyltransferase (MGMT) applied as adjuncts during treatment have been tested in clinical trials [88], [89]. Whilst inhibitors of MGMT may increase the cell killing effects of the alkylating agents, they also enhance the risk of secondary tumors due to the accumulation of mutagenic O6meG residues in DNA (reviewed in [90]). An alternative and less mutagenic strategy to enhance the efficacy of alkylation-based chemotherapy could be to inhibit AAG or ABH2. In support of this proposal, we have recently demonstrated that the cervical carcinoma cell line, HeLa, depleted for AAG by vector-based RNA interference, was sensitised to several alkylating agents, including a 10-fold sensitisation to the chemotherapeutic agent temozolomide [73]. Moreover, here we show that two derivatives of an ovarian carcinoma cell line, A2780, similarly depleted of AAG were also sensitised to temozolomide killing by 5–6-fold (Fig. 6).