A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges

Abstract

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

“…In this work, we have demonstrated that XRCC1 is physically associated with PARP in vivo and in vitro. The DNA repair factor XRCC1 complements the CHO cell lines EM9 and EM-C11, which are hypersensitive to both monofunctional alkylating agents and ␥ rays (50,51,58). In response to genotoxic agents that induce DNA base damage, EM9 and EM-C11 exhibit 10-fold increases in the occurrence of sister chromatid exchanges and severe defects in the rejoining of DNA breaks (19,31,58).…”

“…The DNA repair factor XRCC1 complements the CHO cell lines EM9 and EM-C11, which are hypersensitive to both monofunctional alkylating agents and ␥ rays (50,51,58). In response to genotoxic agents that induce DNA base damage, EM9 and EM-C11 exhibit 10-fold increases in the occurrence of sister chromatid exchanges and severe defects in the rejoining of DNA breaks (19,31,58). Consistent with its proposed role in mammalian BER, XRCC1, which has no demonstrated catalytic activity, is supposed to serve as a scaffold protein during the BER reaction through its interaction with DNA ligase III (7,25,37) and DNA polymerase ␤ (25).…”

Targeting XRCC1 Deficiency in Breast Cancer for Personalized Therapy

“…Zdzienicka, Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus University in Torun (Bydgoszcz, Poland; ref. 25,26). Cells were grown in Ham F-10 media (supplemented with 10% FBS and 1% penicillin/streptomycin; PAA).…”

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

“…49 Wild-type CHO-9 and mutants originating from it are XR-C1-defective for DNA-PK, 50 43-3B defective for ERCC1, which is involved in nucleotide excision repair, 51 and EM-C11 defective for XRCC1 involved in base excision repair. 52 The hamster cells were cultured in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA Laboratories) and penicillin/streptomycin and incubated at 37°C in a humidified atmosphere with 7% CO2. Geneticin (G418 sulfate) was added at a concentration of 500 μg/ ml to cell cultures of V-C8mBAC, VC8+MGMT, Irs1-SF+MGMT, V3 and V3-WT cells.…”