Chloroethylating anticancer drug-induced mutagenesis and its repair in Escherichia coli

Abstract

Background Chloroethylnitrosourea (CENU) derivatives, such as nimustine (ACNU) and carmustine (BCNU), are employed in brain tumor chemotherapy due to their ability to cross the blood-brain barrier. They are thought to suppress tumor development through DNA chloroethylation, followed by the formation of interstrand cross-links (ICLs) that efficiently block replication and transcription. However, the alkylation of DNA and ICLs may trigger genotoxicity, leading to tumor formation as a side effect of the chemotherapeutic treatment. Although the involvement of O 6 -alkylguanine-DNA alkyltransferase (AGT) in repairing chloroethylated guanine ( O 6 -chloroethylguanine) has been reported, the exact lesion responsible for the genotoxicity and the pathway responsible for repairing it remains unclear. Results We examined the mutations induced by ACNU and BCNU using a series of Escherichia coli strains , CC101 to CC111, in which reverse mutations due to each episome from F’101 to F’106 and frameshift mutations due to each episome from F’107 to F’111 could be detected. The mutant frequency increased in E. coli CC102, which can detect a GC to AT mutation. To determine the pathway responsible for repairing the CENU-induced lesions, we compared the frequency of mutations induced by CENU in the wild-type strain to those in the ada, ogt (AGT-deficient) strain, uvrA (nucleotide excision repair (NER)-deficient) strain, mismatch repair (MMR)-deficient strains, and recA (recombination deficient) strain of E. coli CC102. The frequencies of mutations induced by ACNU and BCNU increased in the ada, ogt strain, demonstrating that O 6 -chloroethylguanines were formed, and that a portion was repaired by AGT. Mutation induced by ACNU in NER-deficient strain showed a similar profile to that in AGT-deficient strain, suggesting that an NER and AGT play at the similar efficacy to protect E. coli from mutation induced by ACNU. O 6 -Chloroethylguanine is reported to form ICLs if it is not repaired. We examined the survival rates and the frequencies of mutations induced by ACNU and BCNU in the uvrA strain, the recA strain, as well as a double-deficient strain of CC102. The mutation profile of the double-deficient strain was similar to that of the NER-deficient strain, suggesting that an NER protects E. coli from mutations but not recombination. In addition, cell death was more pronounced in the uvrA, recA double-deficient strain than in the single-deficient strains. ...

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation

“…A series of studies evidenced that the N 1, O 6 -EtG intermediate formed from O 6 -ClEtG via an intramolecular cyclization could undergo an electrophilic attack on the complementary cytosine to form dG-dC cross-link. ,,, To compare the preference of ICLs formation (anticancer effect) and AGT-mediated repair of O 6 -alkylguanines induced by CENUs (drug resistance), we performed calculations on the formation of dG-dC cross-link at the same theoretical level as those in the QM region of the models for the AGT: O 6 -alkylguanine system. The energy data mentioned in the following discussion are all obtained from M2.…”

“…Chloroethylnitrosoureas (CENUs) are an important family of bifunctional alkylating agents with wide applications in the clinical treatment of lymphomas, melanomas, and various solid tumors. As illustrated in Figure , CENU chemotherapies, such as 1,3-bis­(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (me-CCNU), and 3-[(4-amino-2-methyl-5-pyrimidinyl)­methyl]-1-(2-chloroethyl)-1-nitrosourea (ACNU), are unstable in physiological condition and spontaneously undergo decomposition to yield active chloroethylating species. − These active electrophilic reagents are capable of alkylating DNA and further leading to single/double-strand breaks and interstrand cross-links (ICLs). − The formation of DNA ICLs, which interferes with normal DNA replication and transcription by preventing the separation of double strands, is the most cytotoxic lesion and is responsible for the antitumor activities . CENU-induced DNA ICLs occur between the N1 site of guanine and the N3 site of the complementary cytosine resulting in dG-dC cross-link through the supposed mechanism shown in Figure .…”

Assessing genotoxic effects of chemotherapy agents by a robust in vitro assay based on mass spectrometric quantification of γ-H2AX in HepG2 cells

“…As chloroethylnitrosoureas derivatives, nimustine and carmustine are typical chloroethylating agents which can be used in tumor chemotherapy, especially brain tumors due to the capacity to get over the blood-brain barrier ( Nikolova et al, 2012 ; Nikolova et al, 2017 ). These two reagents can combine with guanine N1 on one DNA strand and cytosine N3 on the other strand to form inter-strand crosslinks and prevent DNA replication ( Drabløs et al, 2004 ; Yamada et al, 2019 ). However, carmustine was reported to be six to eight times less cytotoxic than nimustine in cell lines ( Büch and Zeller, 2002 ).…”

The DNA Alkyltransferase Family of DNA Repair Proteins: Common Mechanisms, Diverse Functions

“…The co-localization of AGT with sites of active transcription [41,58,111] also hints at a potential role of AGT in the TC-NER pathway similar to that proposed for ATL on large alkyl lesions in DNA. Previous studies also implicated the NER pathway in the repair of O 6ethylguanine, O 6 -chloroethylguanine, and large branched-chain O 6 -alkylguanines [109,[112][113][114]. Whether or not this is mediated by AGT binding to these lesions and whether the TCor GG-NER pathway is involved has yet to be established.…”