The scid Defect Results in Much Slower Repair of DNA Double-Strand Breaks but Not High Levels of Residual Breaks

Abstract

Severe combined immune deficiency (scid) mice fail to produce mature B and T cells and are sensitive to ionizing radiation. They contain a mutation in the 460-kDa catalytic subunit of the DNA-dependent protein kinase that is involved in both V(D)J rejoining and DNA double-strand break (DSB) repair. The kinetics of DSB rejoining was quantified in both scid cells and the parental C.B-17 cells after three different doses of X irradiation: 3, 7.5 and 10 Gy. Repair of DNA DSBs was determined using pulsed-field gel electrophoresis, Southern hybridization and phosphor image analysis. After X irradiation, the cells were allowed to repair at 37 degrees C for up to 1 h or up to 24 h. The most profound difference between the two cell lines was the greatly reduced rate of the slow component of DSB repair in scid cells. C.B-17 cells repaired most of the damage within 1 h, whereas scid cells required 4 to 6 h to reach a similar level after the same dose. No residual or unrepairable DSBs were detected in either cell line 24 h after doses as high as 10 Gy. The scid cells subjected to two doses of 1.5 Gy separated by increasing amounts of time showed no ability to repair sublethal damage between doses, whereas C.B-17 cells receiving two doses of 3.75 Gy separated by increasing periods did show increased levels of survival. These results indicate that scid cells can repair radiation-induced DNA DSBs, although at a reduced rate, but they lack the ability to undergo repair of sublethal damage.

Low-Dose Metronomic Daily Cyclophosphamide and Weekly Tirapazamine: A Well-Tolerated Combination Regimen with Enhanced Efficacy That Exploits Tumor Hypoxia

“…Pilot experiments in C.B-17 SCID mice with combined LDM cyclophosphamide and tirapazamine showed an unexpected degree of toxicity (as measured by weight loss). This may be due to the DNA damage caused by both cyclophosphamide and tirapazamine coupled with the SCID phenotype that results from a mutation in the gene coding for the catalytic subunit of the DNAdependent protein kinase, implicated in DNA damage repair, particularly the repair of DNA double-strand breaks (39). Indeed, primary skin fibroblasts derived form SCID mice were found to be significantly more sensitive to tirapazamine under normoxic conditions, and similarly sensitive under hypoxic conditions, compared with fibroblasts obtained from NIH Swiss nude mice (data not shown).…”

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

“…In DNA-PKcs de®cient cells, however, the contribution of the fast component is reduced without a measurable change in the half time, and the slow component, acting quasi as a backup, removes a substantially larger proportion of DNA DSBs (*70%). Interestingly, DNA DSB repair proceeds to similar ®nal levels in DNA-PKcs pro®cient and DNA-PKcs de®cient cells Nevaldine et al, 1997;Wang et al, 2000).…”

Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group

“…In higher eukaryotes, on the other hand, mutations in genes involved in NHEJ such as KU70, KU80, DNA-PKcs, and DNA ligase IV, increase cell radiosensitivity to killing and compromise rejoining of IR-induced DNA DSBs (Jackson and Jeggo, 1995;Jeggo, 1998;Lieber et al, 1997;Riballo et al, 1999;Takata et al, 1998;Weaver, 1996) suggesting a signi®cant involvement of NHEJ. Analysis of the kinetics of DNA DSB rejoining in these mutants suggests that NHEJ is represented by the fast component of rejoining (Badie et al, 1995;DiBiase et al, 2000;Iliakis et al, 1992;Kemp et al, 1984;Nevaldine et al, 1997;Okayasu and Iliakis 1993;Ouyang et al, 1997;Wachsberger et al, 1999). Recent studies suggest that some of these mutants are still capable of rejoining the majority of DSBs utilizing the slow component, which now becomes dominant and well de®ned (DiBiase et al, 2000;Nevaldine et al, 1997).…”

“…Analysis of the kinetics of DNA DSB rejoining in these mutants suggests that NHEJ is represented by the fast component of rejoining (Badie et al, 1995;DiBiase et al, 2000;Iliakis et al, 1992;Kemp et al, 1984;Nevaldine et al, 1997;Okayasu and Iliakis 1993;Ouyang et al, 1997;Wachsberger et al, 1999). Recent studies suggest that some of these mutants are still capable of rejoining the majority of DSBs utilizing the slow component, which now becomes dominant and well de®ned (DiBiase et al, 2000;Nevaldine et al, 1997).…”