Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Abstract

C hloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto-and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci , which co-stained with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G 1 but increased significantly in S phase and remained at a high level in G 2 , where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O 6 -chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O 6 -chloroethylguanine-triggered

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

“…2E: note the change of scale of the X axis). The death of MGMT-expressing cells at high dose CCNU treatment is likely the result of N-chloroethylations, which are less toxic than O 6 ClEtG [11]. Again, the expression of eATL in these cells had no impact on cell survival following CCNU treatment (Fig.…”

“…However, induction of cell death following CNUs occurs in an MMR independent manner [9]. Thus, intramolecular rearrangement of the O 6 ClEtG adduct leads ultimately to the formation of an N1-guanine-N3-cytosine interstrand crosslink (ICL) [10], which blocks replication, resulting in collapse of replication forks and the formation of DSBs [11].…”

Integrin αVβ3 silencing sensitizes malignant glioma cells to temozolomide by suppression of homologous recombination repair

“…After washing with PBS, cells were incubated with ice-cold methanol for 10 min at −20°C. After three washing steps with PBS, to avoid unspecific binding, the blocking reagent (PBS + 0.25% Triton 100-X + 10% normal goat serum) was added for 1 h. The Rad51/yH2AX staining and evaluation was conducted as described [56]. After washing, nuclei were either stained with TO-PRO-3 (1:1000, 15 min; only for acquisition on LSM), or treated with 10 μL anti-fade medium (Vectashield) with DAPI (only for acquisition with Metafer system).…”

Assessment of DNA interstrand crosslinks in NIH/3T3 cells induced by Chloroethylnitrosoureas

“…Epidemiological studies have shown that almost 80%-90% of human cancers are caused by the environment [1], being chemical pollutants the most important cancer-causing factors. Nitrosoureas are a class of DNA alkylating-agent anticancer drugs that include Chloroethylnitrosoureas (CENUs), such as 1-((4-amino-2-methyl -5-pyrimidinyl) methyl)-3-(2-chloroethyl)-3-nitrosourea (Nimustine, ACNU), 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine, BCNU), 1-(2-chloroethyl)-3-cyclohexyl-L-nitrosourea (Lomustine, CCNU), 1-(2-chloroethyl)-3-(4-methlycyclohexyl) -1-nitrosourea (Semustine, MeCCNU) and 1-[N-(2-chloroethyl)-N-nitrosourea] ethylphosphonic acid diethyl ester (Fotemustine, FUNU) [2]. Previous studies have indicated that the cytotoxicity of CENUs is related to the formation of DNA interstrand crosslinks (ICLs) [3].…”