Induction and repair of DNA damage in normal and ataxiatelangiectasia skin fibroblasts treated with neocarzinostatin

Abstract

Cells from patients with the hereditary multisystem disorder ataxia-telangiectasia (A-T) are hypersensitive to the cytotoxic action of DNA-breaking agents, such as X-rays, bleomycin and neocarzinostatin (NCS). A defect in the repair of a certain DNA lesion induced by all three agents may underlie this hypersensitivity. This DNA lesion may be a certain type of DNA strand break. Most of the previous experiments done with X-rays and bleomycin failed to show any retardation in the rejoining of DNA strand breaks in A-T cells. However, since both A-T homozygous and heterozygous cells are particularly hypersensitive to NCS, we studied the time course of strand breakage induction and repair in A-T skin fibroblast strains treated with NCS, using the sensitive method of alkaline or neutral elution. A linear dose response was obtained for the induction by NCS of single-strand breaks and double-strand breaks. A-T cells did not respond with a higher initial extent of strand breakage compared with normal cells. NCS is an appropriate agent for studying the kinetics of rejoining strand breaks, due to its rapid action in the cells; this action, which is completed within 2--4 min, was studied by monitoring strand break induction, inhibition of DNA synthesis and decrease in cellular survival. The time course of strand break rejoining found after NCS treatment was very similar to that found following X-irradiation: with both single- and double-strand breaks, a rapid phase of rejoining was first noticed (t 1/2 approximately 5 min for single-strand breaks and 20--25 min for double-strand breaks). This was followed by a second, slow phase that continued for several hours. No difference could be detected between normal and A-T cells either with regard to the time course of rejoining or the fraction of non-rejoined breaks remaining several hours after treatment.

DNA-dependent Protein Kinase and XRCC4-DNA Ligase IV Mobilization in the Cell in Response to DNA Double Strand Breaks

“…In addition, anti-Rad51 antibodies were used as a probe of the homologous recombination route for DSB repair. As opposed to nontreated cells, WCEs from Ncs-treated cells contain ␥-H2AX as expected given the high DNA DSB inducing potency of Ncs (32). In addition, XRCC4 in the WCEs of Ncs-treated cells was detected essentially as a slow migrating form.…”

“…The time course of appearance of this recruitment paralleled that of the DSB-specific induction of H2AX phosphorylation. In addition and for the milder doses of Ncs, the disruption of NHEJ protein recruitment and dephosphorylation of ␥-H2AX occurred concomitantly with a kinetics compatible with the rejoining kinetics of Ncs-induced DSBs generated in normal cells (32). The mobilized NHEJ proteins associate in complexes as demonstrated by their co-immunoprecipitation from the P2 fraction of drug-treated cells (Fig.…”

“…Thus, to remove loosely bound nuclear proteins, we adapted a cellular fractionation procedure based on detergent extraction (30). HCT116 colon carcinoma cells were grown on glass coverslips and either treated or not with the radiomimetic drug Ncs for 1 h. Ncs is a natural enediyne antibiotic that has been shown to produce DSBs with selectivity and efficiency higher than IR (31) and to efficiently induce DBSs and cytotoxicity when applied to cells (32). To optimize DSB production and thus the potential recruitment of repair proteins, Ncs was used at a dose about 10-fold higher than the IC 50 determined on theses cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test after 48 h of culture (data not shown).…”

Cellular Responses to the DNA Strand-scission Enediyne C-1027 Can Be Independent of ATM, ATR, and DNA-PK Kinases

“…Despite differences in chemistry and/or localization of DNA damage between IR and radiomimetic drugs, to the best of our knowledge C-1027 is the only agent able to stimulate p53 Ser-15 phosphorylation and DNA replication inhibition in an ATMϪ cell line. Reports have been published showing that several other radiomimetic compounds (for example, bleomycin and neocarzinostatin) induced radioresistant DNA synthesis and increased cell death in ATM-deficient cells (41,42). Compared with the related enediyne neocarzinostatin, C-1027 independence on ATM activity for stimulating p53 phosphorylation is somewhat unexpected since we have shown recently that both C-1027 and neocarzinostatin induced similar changes in Replication Protein A (RPA) protein status in the 293 human embryonic kidney cell line (43).…”

The Induction and Repair of Double-Strand DNA Breaks in Mammalian Cells as Detected by Neutral Elution

“…Van der Schans et al (1983) showed that the fraction of rapidly repaired dsb induced by neutrons was not significantly different from that after t-irradiation. Consequently, the explanation that the rapidly rejoined dsb could have been induced artificially at ssb sites during the elution procedure, is untenable.…”