Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining.

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase.

scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.

Inactivation of p53 results in high rates of homologous recombination.

Using a plasmid substrate which integrates into the genome, we determined that the rate of homologous recombination was suppressed by p53. Human tumor cell lines, mutant or null for p53 had recombination rates 10000-times greater than primary fibroblasts. When isogenic cell pairs from tumor cells or primary fibroblasts were compared, differing only in one genetic change which inactivated p53, the recombination rate increased > 100-fold. Functional inactivation of p53 by dominant mutant p53, by large T antigen of SV40 virus, by E6 protein of human papilloma virus, or by genetic deletion led to the same result. Our results suggest that p53 suppresses spontaneous homologous recombination, and that p53 is not required for recombination to proceed. The mechanism of recombination suppression may be related to the reported association of p53 with Rad 51, but the functional consequences of this association are not yet established. It is suggested that suppression of homologous recombination is the means by which p53 maintains genetic stability.

p53-null cells are more sensitive to ultraviolet light only in the presence of caffeine.

We have shown previously that p53(-/-) fibroblasts show greater sensitization by caffeine to the lethal effects of ionizing radiation compared with p53(+/+) cells. Recently published data have suggested a possible role of p53 in nucleotide excision repair: an association of p53 and xeroderma pigmentosum group B protein and a greater sensitivity to cisplatin of RKO cells transfected with the E6 protein of human papilloma virus (inactivating p53). We show that p53(+/+) and p53(-/-) cells have equal sensitivity to germicidal UV light (as with ionizing radiation). However, the introduction of 2 mM caffeine led to a sensitization enhancement ratio (at 10% survival) of 1.8 in p53(-/-) cells, but only 1.3 in wild-type (p53+/+) cells. Lower doses of caffeine had less effect, and 0.1 mM caffeine resulted in no detectable sensitization of either cell type to UV light in contrast to X-rays. The differential sensitivity of p53(-/-) cells to X-rays and caffeine was thought to be due to override of the G2-M block to cell cycle progression. In response to UV light, cells accumulate in S phase, and the magnitude of S-phase accumulation was observed to be greater in p53(-/-) cells. Caffeine had little effect on the cell cycle distribution in p53(+/+) cells. However, for p53(-/-) cells, a greater proportion were in S phase after treatment with caffeine, and a complete loss of S-phase delay was observed after UV irradiation. In conclusion, the role of p53 in nucleotide excision repair appears to be of little significance for cell survival. Greater sensitization of p53(-/-) cells to caffeine could be mediated via override of S-phase delay.

High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines.

The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.

Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.

Most drug discovery efforts have focused on finding new DNA-damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor-specific killing by modifying tumor-specific responses to that damage. In this report, we ask whether cells lacking the G1-S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2-M arrest. Mouse embryonic fibroblasts genetically matched to be (+) or (-) p53 and rat embryonic fibroblasts (+) or (-) for wild-type p53 function were irradiated with and without caffeine, a known checkpoint inhibitor. At low doses (500 microM), caffeine caused selective radiosensitization in the p53(-) cells. At this low dose (where no effect was seen in p53(+) cells), the p53(-) cells showed a 50% reduction in the size of the G2-M arrest. At higher doses (2 mM caffeine), where sensitization was seen in both p53(+) and p53(-) cells, the radiosensitization and the G2-M override were more pronounced in the p53(-) cells. The greater caffeine-induced radiosensitization in p53(-) cells suggests that p53, already shown to control the G1-S checkpoint, may also influence aspects of G2-M arrest. These data indicate an opportunity for therapeutic gain by combining DNA-damaging agents with compounds that disrupt G2-M arrest in tumors lacking functional p53.