Cancer Research AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, H.
Right arrow Articles by Iliakis, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, H.
Right arrow Articles by Iliakis, G.
[Cancer Research 61, 270-277, January 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Nonhomologous End-Joining of Ionizing Radiation-induced DNA Double-Stranded Breaks in Human Tumor Cells Deficient in BRCA1 or BRCA21

Huichen Wang, Zhao-Chong Zeng, Tu-Anh Bui, Steven J. DiBiase, Wei Qin, Fen Xia, Simon N. Powell and George Iliakis2

Division of Experimental Radiation Oncology, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 [H. W., Z-C. Z., T-A. B., S. J. D., W. Q., G. I.], and Laboratory of Molecular and Cellular Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129 [F. X., S. N. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the BRCA1 or BRCA2 genes predispose to a wide spectrum of familial cancers. The functions of the proteins encoded by BRCA1 and BRCA2 remain to be elucidated, but their interaction and colocalization with hRAD51 suggest a role in homologous recombination and DNA double-strand break (DSB) repair. The role of BRCA1 and BRCA2 in the rejoining of ionizing radiation (IR)-induced DNA DSBs, which may represent a step in the overall process of repair, remains uncertain because recent reports provide conflicting results. Because elucidation of the role of these proteins in DNA DSB rejoining is important for their functional characterization, we reexamined this end point in cells with mutations in either BRCA1 or BRCA2. We show that two pancreatic carcinoma cell lines known to have either wild-type (BxPC3) or mutant forms (Capan-1) of BRCA2 rejoin IR-induced DNA DSBs to a similar extent following biphasic kinetics characterized by a fast and a slow component. Importantly, inactivation of DNA-dependent protein kinase (DNA-PK) by wortmannin generates similar shifts from the fast to the slow component of rejoining in BRCA2-proficient and BRCA2-deficient cells. This suggests that the functioning of either the fast, DNA-PK-dependent component or the slow, DNA-PK-independent component of rejoining is not affected by mutations in BRCA2. Also, a human breast cancer cell line with mutated BRCA1 shows normal rejoining of IR-induced DNA DSBs and levels of inhibition by wortmannin commensurate with the degree of DNA-PK inhibition. These observations fail to confirm a direct role for BRCA1 or BRCA2 in the rejoining of IR-induced DSBs in the genome of human tumor cells and, as a result, an involvement in nonhomologous end-joining. They are in line with similar observations with mutants deficient in genes implicated in homologous recombination and support the view that the radiosensitivity to killing of cells deficient in BRCA1 or BRCA2 derives from defects in this repair pathway.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence accumulates that the structurally unrelated tumor suppressor genes BRCA1 and BRCA2 are involved in the development of a variety of human neoplasms. Women inheriting defective copies of either BRCA1 or BRCA2 genes have a significantly increased lifetime breast cancer risk. Individuals with BRCA1 mutations also show predisposition to ovarian cancer, whereas individuals with BRCA2 mutations are predisposed to prostate cancer (1, 2, 3, 4) ; BRCA2 mutations have also been found in some pancreatic tumors (5 , 6) .

The functions of BRCA1 and BRCA2 proteins have not been elucidated, but biochemical and cell biological data suggest an involvement in DNA damage response and DNA recombination. It has been reported that BRCA1 and BRCA2 interact and colocalize with the DNA repair protein hRAD51 to nuclear foci after DNA damage and at synaptonemal complexes in meiotic cells (7, 8, 9, 10, 11) . Although BRCA2 interacts directly with hRAD51 through at least two binding sites [one in BRC repeats located in the NH2-terminal end of the region encoded by exon 11 and another in the COOH-terminal region of the protein (8 , 11, 12, 13, 14) ], the interaction with BRCA1 is likely to be indirect and mediated by BRCA2 (9) . BRCA1 also associates with the hRad50-hMre11-p95 DNA repair complex (15) . Cells deficient in BRCA1 are deficient in transcription-coupled repair (16 , 17) and display centrosome amplification and a defective G2-M-phase cell cycle checkpoint (18 , 19) . Other reports identify BRCA1 as a target of the ATM kinase or implicate this protein in DNA damage response and checkpoint activation (20 , 21) . BRCA2 has also been implicated in DNA damage response, and BRCA2-deficient cells are sensitive to DNA-damaging agents, including IR3 (8 , 13 , 22, 23, 24, 25) .

Exposure of cells to IR and certain chemotherapeutic agents leads to the generation of DSBs in the DNA. Unrepaired or misrepaired DNA DSBs can lead to cell killing, mutation induction, gene translocation, and cancer (26, 27, 28, 29) . It is thought that two main pathways exist for the repair of DNA DSB in eukaryotic cells: (a) HRR; and (b) NHEJ. Whereas repair by HRR requires extensive homology and the RAD52 epistasis group of genes (RAD50–55, RAD57, MRE11, and XRS2; reviewed in Ref. 30 ), NHEJ does not require homology and is greatly facilitated by the DNA-PK and the ligase IV/XRCC4 complex (reviewed in Refs. 31 and 32 ). Both pathways for DNA DSB repair have been shown to be active from yeast to humans (31) . However, their relative contribution to the rejoining of DNA DSBs varies widely between higher and lower eukaryotes. Whereas yeast remove the majority of IR-induced DNA DSBs by HRR, cells of higher eukaryotes appear to use predominantly NHEJ for this purpose (32) .

Elucidation of the biochemical characteristics of the mechanisms used to repair DNA DSBs and evaluation of the significance in the shift from HRR to NHEJ in higher eukaryotes are of particular importance for our understanding of genomic stability. The suggested roles of BRCA1 and BRCA2 in these processes may be critical but are only now beginning to be elucidated. In one study, the effect of BRCA1 on homologous and nonhomologous recombination was evaluated by measuring integration in the genome of transfected DNA and repair of a DSB induced by I-Sce-I endonuclease in an integrated construct consisting of two differentially mutated neo genes. The results indicated that mouse cells deficient in BRCA1 exhibit gene-targeting defects and a decrease in HRR of DNA DSBs but show slightly increased nonhomologous integration and proficiency at nonhomologous repair of DNA DSBs (33) . Another study showed no discernible defect in the rejoining of IR-induced DNA DSBs as assayed by pulsed-field gel electrophoresis (17) . On the other hand, recently published data suggest a deficiency in IR-induced DNA DSB rejoining as measured by pulsed-field gel electrophoresis in a human tumor cell line with mutated BRCA1 as compared with an isogenic counterpart generated by stable expression of wild-type BRCA1 (34) .

In cells with BRCA2 mutations, a complete halt was reported in the rejoining of IR-induced DNA DSBs for up to 6 h after irradiation that was invoked to explain the increased radiosensitivity of these cells (22) . However, the magnitude of the reported defect in DNA DSB rejoining far exceeds that of mutants deficient in DNA-PK (31 , 32) , and such a defect would be expected to produce a far greater increase in radiosensitivity.

The above-mentioned results in aggregate suggest a role for BRCA1 and BRCA2 in the ultimate repair of DNA DSBs and, as a result, in cell radiosensitivity to killing. However, it is not clear whether the defect can be localized specifically in the rejoining stage of the DNA DSB repair as evaluated by pulsed-field gel electrophoresis because existing results are either contradictory or difficult to interpret. To contribute to the resolution of this important issue, we designed experiments evaluating DNA DSB rejoining in cells with defects in either BRCA1 or BRCA2. We examined the effect of these mutations on the fast and the slow component of DNA DSB rejoining and evaluated the shift from the fast to the slow component after treatment with wortmannin. Wortmannin is known to inhibit the fast component of DNA DSB rejoining by inhibiting DNA-PK, thus allowing the slow component to become dominant and remove a far greater proportion of DNA DSBs than it does in untreated cells (35) . We reasoned that if BRCA1 or BRCA2 mutations compromise the fast component of NHEJ, and inhibition of DNA-PK produces no additional effect, then it could be argued that the two proteins operate in the same pathway. If, on the other hand, BRCA2 mutant cells show the expected inhibition of the fast component of NHEJ under conditions that inhibit DNA-PK activity, then it could be argued that the two proteins operate in different pathways.

The results indicate that under the experimental conditions used, cells deficient in BRCA1 or BRCA2 rejoin IR-induced DNA DSBs with kinetics and to an extent similar to that of wild-type cells. Furthermore, inactivation of DNA-PK by treatment of cells with wortmannin generates similar levels of inhibition for the fast component of rejoining in cells with mutant or wild-type BRCA1 or BRCA2, particularly when the level of DNA-PK inhibition is taken into consideration. Thus, BRCA1 or BRCA2 mutant cells effectively use DNA-PK-dependent NHEJ to remove DSBs from their genome, and the overall efficiency of this pathway of rejoining is similar to that of wild-type cells. These results support the view that defects in BRCA1 or BRCA2 do not confer a deficiency in the rejoining of IR-induced DNA DSBs. This phenotype is similar to that reported for mutants with defects in genes implicated in HRR, where an increase in radiosensitivity to killing was not accompanied by an obvious defect in DNA DSB rejoining (30) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The Capan-1, Capan-2, and BxPC3 cell lines are derived from human pancreatic carcinomas and were obtained from American Type Culture Collection (Manassas, VA). Capan-1 and HCC1937 cells were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 1.5 grams/liter sodium bicarbonate, 4.5 grams/liter glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 15% fetal bovine serum. BxPC3 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum. Capan-2 cells were grown in MEM supplemented with 10% bovine calf serum. Before beginning repair experiments, the growth characteristics of the cells were investigated in detail, and the results were used to establish procedures for continuous maintenance of the cells in culture, as well as for the preparation of cultures for experiments. Cells were maintained in a phase of nearly logarithmic growth by subculturing every 4 days at an initial concentration of 106 cells/100-mm tissue culture dish. Cells from subculture were also used to set up experiments. For this purpose, 3 x 105 cells were plated per 60-mm dish and allowed to grow for 3 days. At this point, cells reached a density of approximately 106 cells/dish and were irradiated to measure cell survival or the kinetics of DNA DSB rejoining.

Irradiation.
Cells were irradiated using a Pantak X-ray machine operated at 310 kV, 10 mA with a 2 mm Al filter (effective photon energy about 90 kV), at a dose rate of ~2.7 Gy/min. Dosimetry was performed with a Victoreen dosimeter that was used to calibrate an in-field ionization monitor.

Colony-forming Assay.
Cell radiosensitivity to killing was determined by the clonogenic assay. Cells were trypsinized at 37°C immediately after irradiation at room temperature and seeded into 100-mm tissue culture dishes at various densities, aiming at a total of 30–150 colonies/dish. After an incubation period of up to 3 weeks, cells were stained with crystal violet, and colonies of more than 50 cells were counted.

DNA DSB Repair.
Cells for DNA DSB repair experiments were labeled with 0.01 µCi/ml [14C]thymidine plus 2.5 µM cold thymidine for the entire period of growth. When indicated by the experimental protocol, cells were treated with 20–30 µM wortmannin (Sigma) for 1 h before irradiation. Cells were cooled to 4°C before irradiation and irradiated on ice. After irradiation, the medium was replaced with fresh growth medium prewarmed at 40°C (to rapidly restore a temperature of 37°C to the cultures), and cells were returned to the incubator at 37°C to allow for repair. Cells were prepared for DNA DSB analysis at various times thereafter.

It is essential to allow cells to repair DNA DSBs under conditions optimal for growth. We have frequently observed that cells maintained in suspension during repair or cells allowed to repair after embedding in agarose rejoin DNA DSBs more slowly than cells maintained in dishes. After completion of the repair time interval, cells were trypsinized (30 min on ice for the first 4 h and 5 min at 37°C at later times), centrifuged, and resuspended in serum-free medium at a concentration of 6 x 106 cells/ml. This cell suspension was mixed with an equal volume of 1% agarose (InCert agarose; BioWhittaker Molecular Applications), and 3 x 5-mm cylindrical blocks were made containing approximately 1 x 105 cells/block (36) . Blocks were then placed in lysis buffer containing 10 mM Tris (pH 8.0), 50 mM NaCl, 50 mM EDTA, 2% N-lauryl sarcosyl, and 0.1 mg/ml proteinase E and O (Sigma) and incubated at 4°C for at least, 45 min and then incubated at 50°C for 16–18 h. After lysis, agarose blocks were washed for 1 h at 37°C in a buffer containing 10 mM Tris (pH 8.0) and 0.1 M EDTA and then treated for 1 h at 37°C in the same buffer (at pH 7.5) with 0.1 mg/ml RNase A. Cells from identically treated, nonirradiated cultures were also processed at predefined times to determine the signal generated by nonirradiated cells (background). A similar protocol was also used to determine induction of DNA DSBs, except that in this case, cells were embedded in agarose before irradiation on ice and lysed immediately thereafter.

Pulsed-field Gel Electrophoresis.
AFIGE (36) was carried out in 0.5% Seakem-agarose (BioWhittaker Molecular Applications), cast in the presence of 0.5 µg/ml ethidium bromide in 0.5x TBE [45 mM Tris (pH 8.2), 45 mM boric acid, 1 mM EDTA] at 10°C for 40 h. During this time, cycles of 1.25 V/cm for 900 s in the direction of DNA migration alternated with cycles of 5.0 V/cm for 75 s in the reverse direction. Subsequently, gels were dried, exposed to radiation-sensitive screens for 48–96 h, and analyzed by means of a PhosphorImager (Molecular Dynamics) to quantitate DNA damage. For this purpose, the FAR was estimated in irradiated and nonirradiated samples (37) . The FAR measured in nonirradiated cells (background) was subtracted from the results shown with irradiated cells. Gel images were obtained by photographing ethidium bromide-stained gels under UV light or from the PhosphorImager.

Repair kinetics were fitted assuming two exponential components of rejoining according to the equation FAR = Ae-bt + Ce-dt (37) . The first term in the equation was fitted to the fast component of rejoining, and the second term in the equation was fitted to the slow component of rejoining. Fitting was achieved using the nonlinear regression analysis routines of a commercially available software package (SigmaPlot). Parameters A and C describe the amplitudes, and parameters b and d describe the rate constants of the fast and the slow components of rejoining, respectively. From these parameters, the half-times for the rejoining of the fast and the slow components were calculated as t50,fast = ln2/b and t50,slow = ln2/d, respectively. The fraction of DSBs rejoined with fast and slow kinetics was calculated as Ffast = A/(A + C) and Fslow = C/(A + C), respectively. Constrains were occasionally applied during fitting as indicated in the analysis of the individual experiments.

PCR Analysis and DNA Sequencing.
Capan-1 and BxPC3 cell lines were harvested in 10 mM Tris-HCl (pH 7.5), 50 mM EDTA, and 1 mg/ml proteinase K and incubated overnight at 55°C. The cellular DNA was extracted with phenol-chloroform and precipitated by ethanol. PCR of cellular DNA was performed with the BC11-RP (5'-GGGAAGCTTCATAAGTCAGTC-3') and BC11-LP (5'-TTTGTAATGAAGCATCTGATACC-3') primers. PCR products were analyzed on 5% polyacrylamide gel and autosequenced with BC11-RP or BC11-LP primer (ABI-PRISM).

DNA-PK Activity Assay.
Whole cell extracts were prepared by resuspending cells in 3 packed-cell volumes of hypotonic buffer [10 mM HEPES (pH 7.5), 5 mM KCl, 1.5 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT] and breaking the cells with three cycles of freezing at -80°C and thawing at 37°C. Subsequently, the concentration of KCl was adjusted to 500 mM, and the mixture was incubated at 4°C for 30 min on a rotating platform. After a 40-min centrifugation at 4°C, the supernatant was removed and dialyzed [25 mM HEPES (pH 7.5), 100 mM KCl, 1 mM EDTA, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT]. The supernatant of a further centrifugation at 14,000 rpm for 10 min was designated whole cell extract and used to determine DNA-PK activity after measuring protein concentration using the Bradford assay (Bio-Rad). A previously described assay measuring phosphorylation of a p53 peptide was used with slight modifications (38) . Reactions (20 µl) contained 0.2 mM synthetic peptide, 50 mM HEPES·KOH (pH 7.5), 10 mM MgCl2, 0.2 mM EGTA, 1 mM DTT, 50 mM KCl, 10 µg/ml sonicated calf thymus DNA, 5 mM ATP, and 0.2 µCi of [{gamma}-32P]ATP and were incubated at 25°C for 15 min. Extracts or purified proteins were added at a concentration that ensured that the reaction took place in the linear range. The addition of an equal volume of 30% acetic acid with 1 mM ATP stopped the reactions. Twenty µl of this mixture were spotted on phosphocellulose paper and washed extensively with 15% acetic acid; the incorporated activity was counted and used to calculate the kinase activity.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BRCA2 Mutant Cells Are Proficient in DNA DSB Rejoining.
It has been reported previously that Capan-1 cells carry the 6174delT mutation found in the Ashkenazi Jewish population (22 , 39) . This mutation truncates the COOH-terminal half of BRCA2 that contains the hRAD51-interacting domain and may thus compromise the potential of BRCA2 to support homologous recombination and, as a result, probably also DNA DSB repair. Capan-1 cells show loss of heterozygosity at this locus and as a result do not contain a normal copy of the BRCA2 gene (22) . We confirmed the presence of this mutation in our population of cells using PCR analysis. For this purpose, DNA was extracted from Capan-1 and BxPC3 cells, and the region of the mutation was amplified and sequenced. Fig. 1Citation , A—C, shows the amplified fragment and the sequencing data in the region of interest for Capan-1 and BxPC3 cells. The results confirm the expected 6174T deletion in Capan-1 cells.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. PCR sequencing of BRCA2 and cell radiosensitivity to killing in Capan-1 and BxPC3 cell lines. A, a BRCA2 gene fragment was amplified from genomic DNA of Capan-1 (95 bp) and BxPC3 (96 bp) and run on a 6% polyacrylamide gel. A 100-bp marker is shown for comparison. The 1-bp difference between the fragment amplified from Capan-1 and BxPC3 cells is not directly visible under the gel electrophoresis conditions used. B, the PCR products from Capan-1 cells were extracted with phenol-chloroform and used as template for PCR sequencing with primer BC11-LP. C, results as described in B, but for BxPC3 cells. Notice that T at 6174 of BRCA2 is deleted in Capan-1 cells. D, survival curves of exponentially growing Capan-1 and BxPC3 cells. Cells in the exponential phase of growth were irradiated and plated immediately thereafter to measure survival by colony formation. The average and SE from three determinations are shown.

 
To validate the pulsed-field gel electrophoresis conditions used in the present study and obtain information on the shape of the dose-response curve, which is needed for the quantitative analysis of the repair experiments, we evaluated induction of DNA DSBs in Capan-1 and BxPC3 cells. Fig. 2Citation shows the results obtained. The top panel of the figure is a typical gel, and the bottom panel shows the quantitative data obtained from three independent experiments. The results indicate a steady increase in FAR, a measure of the DNA DSBs present, with increasing dose of IR for both cell lines. A straight line for doses up to 40 Gy can approximate the increase in FAR. There are no significant differences in the yields of DNA DSBs between Capan-1 and BxPC3 cells, as indicated by the similar increase in FAR as a function of radiation dose. Because induction of DNA DSBs as a function of radiation dose is considered linear (28) , the nearly linear relationship between FAR and radiation dose in the range between 0 and 40 Gy also implies a nearly linear relationship between FAR and the DNA DSBs present. As a result, DNA DSB rejoining can be evaluated directly from FAR versus time plots, obviating the corrections otherwise required when the relationship between FAR and radiation dose deviates significantly from linearity (40) .



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Induction of DNA DSBs as a function of radiation dose in exponentially growing Capan-1 and BxPC3 cells. Cells were trypsinized, embedded in agarose blocks, and exposed to various doses of X-rays while maintained on ice. The amount of DNA DSBs present was measured by AFIGE and is expressed as FAR. A shows typical gels, and B shows the results obtained by quantitating gels from three to four experiments. The mean and the SE are plotted. The value of FAR measured in nonirradiated cells was between 3–9% and was subtracted from the FAR values shown. The line represents the best linear fit of Capan-1 results.

 
We systematically studied rejoining of DNA DSBs in Capan-1 and BxPC3 cells after exposure to 40 Gy of X-rays under optimal conditions of growth. The experiments allowed prolonged postirradiation incubation times to assess the ultimate fate of IR-induced DNA DSBs in the genome of BRCA2-deficient cells. Fig. 3ACitation shows the results obtained in experiments where rejoining in Capan-1 cells was followed for up to 10 h postirradiation. Results obtained with irradiated and nonirradiated samples are shown in the figure. Rejoining of DNA DSBs, as manifested by the reduction in FAR, is practically complete at 6 h. The solid line in Fig. 3ACitation was obtained by fitting the results to the sum of two exponential functions as outlined in "Material and Methods." From this fitting, half times of 46 min and 11.5 h were calculated for the fast and the slow components of rejoining, respectively. As indicated in Table 1Citation , 74% of IR-induced DNA DSBs are removed with fast kinetics, and the remaining 26% are removed with slow kinetics. Thus, a fast component dominates DNA DSB rejoining in Capan-1 cells and removes three-fourths of all breaks.



View larger version (52K):
[in this window]
[in a new window]
  		Fig. 3. A, rejoining of IR-induced DNA DSB in exponentially growing Capan-1 cells in the presence (•) or absence ({circ}) of 20 µM wortmannin. For rejoining in the absence of wortmannin, cells were exposed to 40 Gy of X-rays as described in "Materials and Methods" and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. For rejoining in the presence of wortmannin, cells were exposed to the drug for 1 h, irradiated, and returned to 37°C. At various times thereafter, cells were trypsinized and prepared for AFIGE. a shows typical gels, and b shows the results obtained by quantitating three to five gels. The mean and the SE are plotted. The value of FAR measured in nonirradiated cells has been subtracted from all data points. The lines represent fitting to a double exponential as outlined in detail in the text. See Table 1Citation for the parameters derived from this fitting. The figure also shows the results obtained with nonirradiated samples to determine the background. B, rejoining of DNA DSBs in exponentially growing BxPC3 cells. Other details were as described in A.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Parameters of DNA DSB rejoining obtained by fitting to a double exponential formalism as outlined in the text

The results obtained with untreated cells and with cells treated with wortmannin are shown. t50, fast is the half time of the initial fast component of rejoining; t50, slow is the half time of the final slow component of rejoining; % DSBs, fast indicates the fraction of DNA DSBs rejoined with fast kinetics, and % DSBs, slow indicates the percentage of DNA DSBs rejoined with slow kinetics.

 
To examine whether DNA DSB rejoining measured under these conditions utilized the DNA-PK-dependent pathway of NHEJ, we tested Capan-1 cells treated with 20 µM wortmannin. Wortmannin is a potent inhibitor of phosphatidylinositol 3'-kinase, but at higher concentrations it also inhibits other members of the family, such as DNA-PK, and has a pronounced effect on DNA DSB rejoining (35 , 41, 42, 43, 44, 45) . It has recently been shown that wortmannin inhibits the fast component of rejoining of DNA DSBs, mainly by inhibiting DNA-PK activity, and that it is only marginally active in DNA-PKcs-deficient cell lines (35 , 43) . The results obtained are included in Fig. 3ACitation . This data can be adequately fitted using the half times calculated for untreated cells ({circ}). The quality of the fitting indicates that wortmannin does not significantly change the half-times of either the fast or slow component of DNA DSB rejoining but reduces the proportion of DNA DSBs rejoined with fast kinetics. According to this analysis, 23% of the breaks are rejoined with fast kinetics and 77% are rejoined with slow kinetics in Capan-1 cells treated with 20 µM wortmannin (see Table 1Citation ). This response is similar to that observed with other cell lines and suggests that Capan-1 cells rejoin DNA DSB with characteristics similar to those reported for other human tumor cell lines without defects in BRCA2 (35) .

To confirm this observation, we measured rejoining of DNA DSBs under similar experimental conditions in BxPC3 cells that have wild-type BRCA2 (see Fig. 1Citation ), and the results obtained are shown in Fig. 3BCitation . As with Capan-1 cells, active rejoining is observed, which leads to nearly complete removal of DNA DSBs 6 h after irradiation. Notably, the data can be adequately fitted with the repair half times calculated for Capan-1 cells (solid line) and allow us to estimate that 75% and 25% of the DSBs are rejoined with fast and slow kinetics, respectively (see Table 1Citation ). Wortmannin inhibits the fast component of DNA DSB rejoining in BxPC3 cells as well. The qualitative characteristics of the inhibition are similar to those observed in Capan-1 cells, and fitting with the same repair half times allows us to estimate that 33% and 67% of the breaks are rejoined with fast and slow kinetics, respectively (see Table 1Citation ). Overall, the data obtained with BxPC3 cells are practically superimposable with those obtained with Capan-1 cells and suggest that within the experimental uncertainties, the two cell lines rejoin DNA DSBs with indistinguishable kinetics. This is true for results obtained with untreated cells as well as those obtained with cells treated with wortmannin, suggesting that the contribution of DNA-PK-dependent NHEJ to the overall rejoining reaction is similar in the two cell lines. A second cell line known to also contain a wild-type BRCA2 gene (Capan-2) also gave similar results (data not shown).

The lack of identifiable differences in the kinetics of DNA DSB rejoining between Capan-1 and BxPC3 cells prompted us to examine radiosensitivity to killing. Fig. 1DCitation shows the results obtained. As reported previously (22) , Capan-1 cells are radiosensitive to killing when compared with BxPC3 cells. Thus, the increased radiosensitivity of Capan-1 cells is not associated with a defect in the rejoining of IR-induced DNA DSBs.

BRCA1 Mutant Cells Are Proficient in DNA DSB Rejoining.
We examined rejoining of DNA DSBs in HCC1937, a cell line established from a human breast carcinoma known to be homozygous for the BRCA1 5382 insC mutation that eliminates the BRCT domain of the protein (46) . Cells in the exponential phase of growth were irradiated, and rejoining of DNA DSBs was determined in the presence or absence of wortmannin. Fig. 4Citation shows the results obtained. The top panel shows typical gels, whereas the bottom panel shows the results obtained by quantitation of different gels from independent experiments. The increase in FAR as a function of radiation dose (top gel in the top panel and the inset in the bottom panel) is similar to that observed with Capan-1 cells, although it shows a slightly stronger bending trend for doses above 30 Gy. Consideration of the bending of the dose-response curve in the analysis of repair results did not significantly alter their quantitation. Therefore, DNA DSB rejoining is presented in FAR versus time plots as seen for Capan-1 and BxPC3 cells.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4. Induction and rejoining of DNA DSB in irradiated HCC1937 cells. A shows typical gels, whereas B shows the quantitation from several gels. The inset in B shows a dose-response curve obtained by quantitating gels (three gels) similar to those shown in A (top gel). The kinetics of DNA DSB rejoining is shown in B (from five gels), and a typical gel is shown in A (bottom three gels). The line drawn through the repair data represents fitting to the sum of two exponentials as outlined in the text. In this set, cells were allowed to repair in the presence of 20 or 30 µM wortmannin. Other details were as described in the Fig. 3Citation legend.

 
Rejoining of DNA DSBs proceeds efficiently in HCC1937 cells in the absence of wortmannin (second gel in the top panel and • in the bottom panel). Fitting the data to the sum of two exponential functions gives 13 min and 3.1 h for the half times of the fast and slow component of DNA DSB rejoining, respectively. These values are significantly shorter than those measured in Capan-1 or BxPC3 cells but are within the range usually measured in mammalian cells; they are probably a reflection of the overall metabolism of the HCC1937 cells. Similar to the results obtained with Capan-1 and BxPC3 cells, 79% of the DNA DSBs are removed by the fast component of rejoining, and 21% of the DNA DSBs are removed by the slow component of rejoining (Table 1)Citation .

Wortmannin at 20 µM significantly inhibits DNA DSB rejoining in HCC1937 cells. Fitting of the results to the sum of two exponential functions gives 13 min and 13.2 h for the kinetics of the fast and slow component of rejoining. Thus, in these cells, wortmannin modifies the kinetics of rejoining of the slow component, suggesting that it is not the typical DNA-PK-independent component. It is possible that the slow, DNA-PK-independent component of DNA DSB rejoining is not measurable in untreated HCC1937 cells. However, its operation becomes clearly obvious after treatment with wortmannin. From this fitting, it can also be estimated that 52% of the breaks are rejoined with fast kinetics, whereas 48% are rejoined with slow kinetics (see Table 1Citation ). Compared with Capan-1 and BxPC3 cells, HCC1937 cells appear to be less responsive in shifting rejoining of DNA DSBs from the fast to the slow component after treatment with 20 µM wortmannin. To evaluate whether this effect is due to a reduced effectiveness of wortmannin in HCC1937 cells, we carried out an experiment using 30 µM wortmannin. The results obtained (Fig. 4)Citation could be fitted with repair half times similar to those estimated for 20 µM wortmannin, but now only 40% of the DNA DSBs are removed by the fast component of rejoining, with the remaining 60% being removed by the slow component of rejoining.

Whereas the relatively large contribution of the fast component of rejoining after treatment with wortmannin may be misleading and may derive from the fact that in these cells, the slow component was DNA-PK sensitive (see above), we also inquired whether reduced efficacy of wortmannin in inhibiting DNA-PK in HCC1937 cells contributes to this effect. For this purpose, we measured residual DNA-PK activity in extracts of Capan-1, BxPC3, and HCC1937 cells treated with different concentrations of wortmannin and compared the results with those obtained with HeLa cells, which were included as a control. For these experiments, whole cell extracts are prepared from nonirradiated, wortmannin-treated cells and assayed after extensive dialysis. DNA-PK activity is measured as described in "Material and Methods." Fig. 5Citation shows the results obtained. It is evident that the same concentration of wortmannin produced in HeLa, BxPC3, or Capan-1 cells approximately twice the inhibition observed in HCC1937 cells. This result suggests that the reduced efficacy of wortmannin in inhibiting DNA DSB rejoining in HCC1937 cells partly reflects the reduced ability of the drug to inhibit DNA-PK and other wortmannin-sensitive kinases in the treated cells and that higher drug concentrations are needed to achieve the same effect. The reasons for this variation in the efficacy of wortmannin are not known, but it is possible that they derive from differences in the ATP content of the cells. Indeed, wortmannin-induced inhibition of DNA-PK is known to be competitive with ATP (47) .



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 5. Inhibition of DNA-PK activity achieved by a 2-h exposure of exponentially growing cells to the indicated concentrations of wortmannin. After treatment, cells were collected by trypsinization, and whole cell extracts were prepared. Residual activity was determined as outlined under "Materials and Methods." The results have been normalized to the activity measured in each cell line in the absence of wortmannin. The absolute activity of DNA-PK in Capan-1 and BxPC3 cells was approximately 30% higher than that in HCC1937 cells, and the absolute activity of DNA-PK in HeLa cells was approximately 30% higher than that in Capan-1 cells. Activity was below 5% in M059-J cells known to be deficient in DNA-PK (data not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
BRCA1 and DNA DSB Rejoining.
The results presented above indicate that cells with mutant BRCA1 rejoin DNA DSBs with efficiency and to an extent similar to that observed in cells expressing endogenous wild-type BRCA1. This observation is in line with a recent report in which HCC1937 cells were tested for DNA DSB rejoining using pulsed-field gel electrophoresis technology similar to that used in the present study (17) . Because rejoining of radiation-induced DNA DSBs is thought to largely reflect NHEJ (31 , 32 , 35) , our results and those published previously (17) are in agreement with the observation that nonhomologous integration of transfected DNA in the genome remains unchanged in mouse embryo fibroblasts from BRCA1 knockout mice, as compared with their isogenic counterparts expressing wild-type BRCA1 (33) . Also, NHEJ of DSBs induced by I-Sce-I endonuclease in an integrated construct consisting of two differentially mutated neo genes is similar in BRCA1-/- and BRCA1+/- cells (33) . These results, in aggregate, are compatible with the view that BRCA1 is not directly involved in the closing of IR-induced DNA DSBs and suggest that the increased radiosensitivity and genomic instability of cells with mutant BRCA1 derive from associated defects in other repair pathways.

The above-mentioned conclusion, however, is challenged by a recent report with HCC1937 cells (34) . In the latter study, an elegant methodology is used to generate cells isogenic to HCC1937, stably expressing BRCA1 at levels comparable to those of wild-type human cells, and responses of the corrected cells are compared with those of control HCC1937 cells generated by infection with empty constructs. The results indicate that corrected cells sustain less DNA DSBs than control HCC1937 cells and rejoin IR-induced DNA DSBs faster and more completely. On the basis of these observations, it was proposed that rejoining of IR-induced DNA DSBs is defective in cells with mutated BRCA1 and that this deficiency explains in part the increased radiosensitivity of these cells (34) .

The reasons for the discrepancy between these results and those presented here, as well as those published previously (17 , 33) , are not clear. However, it may be indicative that the difference derives from incomplete rejoining of DNA DSBs in the derivatives of HCC1937 cells generated by infection with empty retroviral expression vectors and used as controls in the evaluation of the response of the corrected cells. Thus, whereas the results in Fig. 4Citation and those of Abbott et al. (17) indicate complete rejoining within 6 h of DNA DSBs induced by 40 and 10 Gy of X-rays, respectively, the results obtained with infected HCC1937 cells indicate 20–30% unrejoined breaks after exposure to 8 Gy, even 24 h after irradiation. Because results with nonmanipulated HCC1937 cells are not shown in this study, it remains possible that retroviral infection and BRCA1 expression somehow affect the response of cells to DNA DSB rejoining.

Incubation of HCC1937 cells with wortmannin causes an inhibition of DNA DSB rejoining, which correlates with the observed inhibition in DNA-PK activity (Figs. 4Citation and 5)Citation . These results indicate that HCC1937 cells use the DNA-PK-dependent NHEJ pathway for the fast rejoining of radiation-induced DNA DSBs, despite their mutant BRCA1 status. The contribution of this pathway to the overall rejoining is similar to that of wild-type cells (see Table 1Citation and Ref. 35 ), suggesting that defects in BRCA1 do not compromise its operation.

BRCA2 and DNA DSB Rejoining.
The results presented in Fig. 3Citation indicate that cells with mutant BRCA2 rejoin IR-induced DNA DSBs with efficiency and to an extent similar to that observed in BRCA2-proficient cells. Furthermore, incubation of cells with wortmannin inhibits the fast component of DNA DSB rejoining to a similar extent in cells with mutant BRCA2 or in cells expressing wild-type protein. These observations suggest that mutation in BRCA2 is not associated with a defect in DNA DSB rejoining either under normal conditions or under conditions compromising NHEJ by inhibiting DNA-PK. This conclusion differs from that derived in earlier experiments using the same cell lines (22) . Whereas the reasons for the difference are not clear, it is possible that the reduced rejoining reported earlier is due, at least in part, to the fact that cells were allowed to repair while suspended in agarose. In the experiments presented here, cells were maintained after irradiation under optimal growth conditions for the entire period of incubation and prepared for gel electrophoresis only after completion of the repair time interval. We have observed that cells allowed to rejoin DNA DSBs while embedded in agarose show slower kinetics and increased DNA degradation compared with cells maintained for repair as a monolayer. The magnitude of this effect varies between different cell lines and may be large in cells defective in BRCA2. The observations here are further supported by recent data indicating normal levels of random integration of transfected DNA in cells with mutant BRCA2 and similar kinetics of IR-induced DNA DSB rejoining in Capan-1 cells and a corrected cell line generated by expressing wild-type BRCA2.4 The same study provides strong evidence for a specific regulation of homologous recombination by BRCA2 (see the text below). It is hypothesized that this function maintains genomic integrity and suppresses tumor development in proliferating cells.4

Repair Defects of BRCA1- and BRCA2-deficient Cells.
The above-mentioned results, in aggregate, suggest that BRCA1 and BRCA2 are not directly involved in the rejoining of DNA DSBs as assayed by pulsed-field gel electrophoresis in irradiated cells. The similar inhibition by wortmannin further suggests that this holds true both for the fast, DNA-PK-dependent component and the slow, DNA-PK-independent component of rejoining (35) . Therefore, the increased radiosensitivity of cells with mutations in these genes may derive from defects conferred to other repair pathways. Cells with mutant BRCA1 are defective in transcription coupled repair, and expression of wild-type BRCA1 corrects this deficiency (16 , 17) . Furthermore, both BRCA1 and BRCA2 have been implicated in HRR (see "Introduction"). If BRCA1 and BRCA2 were involved predominantly in HRR, a defect in DNA DSB rejoining may not be expected because several cell lines with documented defects in genes involved in HRR show normal ability in rejoining IR-induced DNA DSBs. This is true for XRCC2 and XRCC3 mutants, as well as for cells with mutations in other homologues (or paralogues) of Rad51 (reviewed in Ref. 30 ). Similar results were also recently obtained in our laboratory using knockout mutants of DT40 cells with defects in various genes of the HRR pathway (data not shown). Thus, a lack of involvement of BRCA1 and BRCA2 in NHEJ is compatible with a role in HRR, which may also explain the increased radiosensitivity of the mutants to killing. Indeed, cells with defects in components of the HRR pathway are frequently radiosensitive to killing despite the lack of obvious deficiencies in DNA DSB rejoining (30) . This is in line with recent observations suggesting a specific defect in HRR for BRCA2-deficient cells.4

Capan-1 cells are more radiosensitive to killing than BxPC3 cells, confirming previous reports with the same cells and other experiments suggesting that mutation in BRCA2 confers an increase in radiosensitivity (see "Introduction").

In summary, our pulsed-field gel electrophoresis results failed to demonstrate a role for either BRCA1 or BRCA2 in the rejoining of DNA DSBs induced by IR in the genome of human cells. This conclusion is in line with results obtained by other investigators using similar or complementary methodologies. It will be instructive to investigate whether the reduced rejoining of DNA DSBs observed in some reports with cells defective in either BRCA1 or BRCA2 reflects the conditions used rather than the mutational status of the genes. Resolution of this issue will clarify the direction of future research and should help the elucidation of the functions of the BRCA1 and BRCA2 genes in normal cells.


   ACKNOWLEDGMENTS
 
We thank Nancy Mott for help in preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants CA42026 and P30-CA56036 awarded from the NIH, DHHS. Back

2 To whom requests for reprints should be addressed, at Thomas Jefferson University, Department of Radiation Oncology, Thompson Building, Room B-1, 1020 Sansom Street, Philadelphia, PA 19107. Phone: (215) 955-6473; Fax: (215) 955-2052; E-mail: George.Iliakis{at}mail.tju.edu Back

3 The abbreviations used are: IR, ionizing radiation; DSB, double-strand break; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase, catalytic subunit; NHEJ, nonhomologous end-joining; HRR, homologous recombination repair; AFIGE, asymmetric field inversion gel electrophoresis; FAR, fraction of activity released from the well into the lane. Back

4 F. Xia, D. G. Taghian, K. M. McDonough, J. S. DeFrank, Z-C. Zeng, H. Willers, G. Iliakis, and S. N. Powell. Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal non-homologous end-joining, manuscript in preparation. Back

5 Xia et al., in press. Back

Received 7/ 7/00. Accepted 11/ 1/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wooster R., Bignell G., Lancaster J., Swift S., Seal S., Mangion J., Collins N., Gregory S., Gumbs C., Micklem G. Identification of the breast cancer susceptibility gene BRCA2. Nature (Lond.), 378: 789-792, 1995.[Medline]
  2. Tavtigian S. V., Simard J., Rommens J., Couch F., Shattuck-Eidens D., Neuhausen S., Merajver S., Thorlacius S., Offit K., Stoppa-Lyonnet D., Belanger C., Bell R., Berry S., Bogden R., Chen Q., Davis T., Dumont M., Frye C., Hattier T., Jammulapati S., Janecki T., Jiang P., Kehrer R., Leblanc J. F., Goldgar D. E., et al The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat. Genet., 12: 333-337, 1996.[Medline]
  3. Teng D. H., Bogden R., Mitchell J., Baumgard M., Bell R., Berry S., Davis T., Ha P. C., Kehrer R., Jammulapati S., Chen Q., Offit K., Skolnick M. H., Tavtigian S. V., Jhanwar S., Swedlund B., Wong A. K., Kamb A. Low incidence of BRCA2 mutations in breast carcinoma and other cancers Nat. Genet., 13: 241-244, 1996.
  4. Miki Y., Swensen J., Shattuck-Eidens D., Futreal P. A., Harshman K., Tavtigian S., Liu Q., Cochran C., Bennett L. M., Ding W., et al A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science (Washington DC), 266: 66-71, 1994.[Abstract/Free Full Text]
  5. Goggins M., Schutte M., Lu J., Moskaluk C. A., Weinstein C. L., Petersen G. M., Yeo C. J., Jackson C. E., Lynch H. T., Hruban R. H., Kern S. E. Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas. Cancer Res., 56: 5360-5364, 1996.[Abstract/Free Full Text]
  6. Thorlacius S., Olafsdottir G., Tryggvadottir L., Neuhausen S., Jonasson J. G., Tavtigian S. V., Tulinius H., Ogmundsdottir H. M., Eyfjord J. E. A single BRCA2 mutation in male and female breast cancer families from Iceland with varied cancer phenotypes. Nat. Genet., 13: 117-119, 1996.[Medline]
  7. Scully R., Chen J., Plug A., Xiao Y., Weaver D., Feunteun J., Ashley T., Livingston D. M. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88: 265-275, 1997.[Medline]
  8. Chen P. L., Chen C. F., Chen Y., Xiao J., Sharp Z. D., Lee W. H. The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. Proc. Natl. Acad. Sci. USA, 95: 5287-5292, 1998.[Abstract/Free Full Text]
  9. Chen J., Silver D. P., Walpita D., Cantor S. B., Gazdar A. F., Tomlinson G., Couch F. J., Weber B. L., Ashley T., Livingston D. M., Scully R. Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2: 317-328, 1998.[Medline]
  10. Chen J. J., Silver D., Cantor S., Livingston D. M., Scully R. BRCA1, BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res., 59: 1752s-1756s, 1999.
  11. Wong A. K. C., Pero R., Ormonde P. A., Tavtigian S. V., Bartel P. L. RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene brca2. J. Biol. Chem., 272: 31941-31944, 1997.[Abstract/Free Full Text]
  12. Mizuta R., LaSalle J. M., Cheng H. L., Shinohara A., Ogawa H., Copeland N., Jenkins N. A., Lalande M., Alt F. W. RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein Proc. Natl. Acad. Sci. USA, 94: 6927-6932, 1997.[Abstract/Free Full Text]
  13. Sharan S. K., Morimatsu M., Albrecht U., Lim D. S., Regel E., Dinh C., Sands A., Eichele G., Hasty P., Bradley A. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature (Lond.), 386: 804-810, 1997.[Medline]
  14. Chen C-F., Chen P-L., Zhong Q., Sharp Z. D., Lee W-H. Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G2/M checkpoint control. J. Biol. Chem., 274: 32931-32935, 1999.[Abstract/Free Full Text]
  15. Zhong Q., Chen C. F., Li S., Chen Y., Wang C. C., Xiao J., Chen P. L., Sharp Z. D., Lee W. H. Association of BRCA1 with the hRad50-hMre11–p95 complex and the DNA damage response. Science (Washington DC), 285: 747-750, 1999.[Abstract/Free Full Text]
  16. Gowen L. C., Avrutskaya A. V., Latour A. M., Koller B. H., Leadon S. A. BRCA1 required for transcription-coupled repair of oxidative DNA damage. Science (Washington DC), 281: 1009-1012, 1998.[Abstract/Free Full Text]
  17. Abbott D. W., Thompson M. E., Robinson-Benion C., Tomlinson G., Jensen R. A., Holt J. T. BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J. Biol. Chem., 274: 18808-18812, 1999.[Abstract/Free Full Text]
  18. Xu X., Weaver Z., Linke S. P., Li C., Gotay J., Wang X. W., Harris C. C., Ried T., Deng C. X. Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol. Cell, 3: 389-395, 1999.[Medline]
  19. Larson J. S., Tonkinson J. L., Lai M. T. A BRCA1 mutant alters G2-M cell cycle control in human mammary epithelial cells. Cancer Res., 57: 3351-3355, 1997.[Abstract/Free Full Text]
  20. Lee J-S., Collins K. M., Brown A. L., Lee C-H., Chung J. H. hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature (Lond.), 404: 201-204, 2000.[Medline]
  21. Somasundaram K., Zhang H., Zeng Y. X., Houvras Y., Peng Y., Wu G. S., Licht J. D., Weber B. L., El-Deiry W. S. Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1. Nature (Lond.), 389: 187-190, 1997.[Medline]
  22. Abbott D. W., Freeman M. L., Holt J. T. Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells. J. Natl. Cancer Inst. (Bethesda), 90: 978-985, 1998.[Abstract/Free Full Text]
  23. Connor F., Bertwistle D., Mee P. J., Ross G. M., Swift S., Grigorieva E., Tybulewicz V. L., Ashworth A. Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation. Nat. Genet., 17: 423-430, 1997.[Medline]
  24. Patel K. J., Vu V. P., Lee H., Corcoran A., Thistlethwaite F. C., Evans M. J., Colledge W. H., Friedman L. S., Ponder B. A., Venkitaraman A. R. Involvement of Brca2 in DNA repair. Mol. Cell, 1: 347-357, 1998.[Medline]
  25. Morimatsu M., Donoho G., Hasty P. Cells deleted for BRCA2 COOH terminus exhibit hypersensitivity to {gamma}-radiation and premature senescence. Cancer Res., 58: 3441-3447, 1998.[Abstract/Free Full Text]
  26. Iliakis G. The role of DNA double strand breaks in ionizing radiation induced killing of eukaryotic cells. Bioessays., 13: 641-648, 1991.[Medline]
  27. Ward J. F. Biochemistry of DNA lesions. Radiat. Res., 104: S103-S111, 1985.
  28. Frankenberg-Schwager M. Review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation. Radiother. Oncol., 14: 307-320, 1989.[Medline]
  29. Ward J. F. Radiation mutagenesis: the initial DNA lesions responsible. Radiat. Res., 142: 362-368, 1995.[Medline]
  30. Thompson L. H., Schild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie (Paris), 81: 87-105, 1999.[Medline]
  31. Smith G. C. M., Jackson S. P. The DNA-dependent protein kinase. Genes Dev., 13: 916-934, 1999.[Free Full Text]
  32. Jeggo P. A. DNA breakage and repair. Adv. Genet., 38: 186-218, 1998.
  33. Moynahan M. E., Chiu J. W., Koller B. H., Jasin M. Brca1 controls homology-directed DNA repair. Mol. Cell, 4: 511-518, 1999.[Medline]
  34. Scully R., Ganesan S., Vlasakova K., Chen J., Socolovsky M., Livingston D. M. Genetic analysis of BRCA1 function in a defined tumor cell line. Mol. Cell, 4: 1093-1099, 1999.[Medline]
  35. DiBiase S. J., Zeng Z-C., Chen R., Hyslop T., Curran W. J. J., Iliakis G. DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. Cancer Res., 60: 1245-1253, 2000.[Abstract/Free Full Text]
  36. Iliakis G., Metzger L., Denko N., Stamato T. D. Detection of DNA double strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis. Int. J. Radiat. Biol., 59: 321-341, 1991.[Medline]
  37. Metzger L., Iliakis G. Kinetics of DNA double strand breaks throughout the cell cycle as assayed by pulsed field gel electrophoresis in CHO cells. Int. J. Radiat. Biol., 59: 1325-1339, 1991.[Medline]
  38. Cheong N., Perrault A. R., Wang H., Wachsberger P., Mammen P., Jackson I., Iliakis G. DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro. Int. J. Radiat. Biol., 75: 67-81, 1999.[Medline]
  39. Neuhausen S., Gilewski T., Norton L., Tran T., McGuire P., Swensen J., Hampel H., Borgen P., Brown K., Skolnick M., Shattuck-Eidens D., Jhanwar S., Goldgar D., Offit K. Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish women affected by breast cancer. Nat. Genet., 13: 126-128, 1996.[Medline]
  40. Wachsberger P. R., Li W-H., Guo M., Chen D., Cheong N., Ling C. C., Iliakis G. Rejoining of DNA double strand breaks in Ku80-deficient mouse fibroblasts. Radiat. Res., 151: 398-407, 1999.[Medline]
  41. Boulton S., Kyle S., Yalcintepe L., Durkacz B. W. Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells. Carcinogenesis (Lond.), 17: 2285-2290, 1996.[Abstract/Free Full Text]
  42. Okayasu R., Suetomi K., Ullrich R. L. Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells. Radiat. Res., 149: 440-445, 1998.[Medline]
  43. Chernikova S. B., Wells R. L., Elkind M. M. Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks. Radiat. Res., 151: 159-166, 1999.[Medline]
  44. Rosenzweig K. E., Youmell M. B., Palayoor S. T., Price B. D. Radiosensitization of human tumor cells by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. Clin. Cancer Res., 3: 1149-1156, 1997.[Abstract]
  45. Price B. D., Youmell M. B. The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. Cancer Res., 56: 246-250, 1996.[Abstract/Free Full Text]
  46. Tomlinson G. E., Chen T. T., Stastny V. A., Virmani A. K., Spillman M. A., Tonk V., Blum J. L., Schneider N. R., Wistuba I. I., Shay J. W., Minna J. D., Gazdar A. F. Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier. Cancer Res., 58: 3237-3242, 1998.[Abstract/Free Full Text]
  47. Wymann M. P., Bulgarelli-Leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol. Cell. Biol., 16: 1722-1733, 1996.[Abstract]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
H.-C. Wang, W.-C. Chou, S.-Y. Shieh, and C.-Y. Shen
Ataxia Telangiectasia Mutated and Checkpoint Kinase 2 Regulate BRCA1 to Promote the Fidelity of DNA End-Joining
Cancer Res., February 1, 2006; 66(3): 1391 - 1400.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Zhuang, J. Zhang, H. Willers, H. Wang, J. H. Chung, D. C. van Gent, D. E. Hallahan, S. N. Powell, and F. Xia
Checkpoint Kinase 2-Mediated Phosphorylation of BRCA1 Regulates the Fidelity of Nonhomologous End-Joining
Cancer Res., February 1, 2006; 66(3): 1401 - 1408.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S.-H. Lee, M. Oshige, S. T. Durant, K. K. Rasila, E. A. Williamson, H. Ramsey, L. Kwan, J. A. Nickoloff, and R. Hromas
The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair
PNAS, December 13, 2005; 102(50): 18075 - 18080.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
E. Honrado, A. Osorio, J. Palacios, R. L. Milne, L. Sanchez, O. Diez, A. Cazorla, K. Syrjakoski, D. Huntsman, P. Heikkila, et al.
Immunohistochemical Expression of DNA Repair Proteins in Familial Breast Cancer Differentiate BRCA2-Associated Tumors
J. Clin. Oncol., October 20, 2005; 23(30): 7503 - 7511.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
J. Zhang and S. N. Powell
The Role of the BRCA1 Tumor Suppressor in DNA Double-Strand Break Repair
Mol. Cancer Res., October 1, 2005; 3(10): 531 - 539.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
R. J. Romeijn, M. M. Gorski, M. A. van Schie, J. N. Noordermeer, L. H. Mullenders, W. Ferro, and A. Pastink
Lig4 and Rad54 Are Required for Repair of DNA Double-Strand Breaks Induced by P-Element Excision in Drosophila
Genetics, February 1, 2005; 169(2): 795 - 806.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
W. D. Block, Y. Yu, D. Merkle, J. L. Gifford, Q. Ding, K. Meek, and S. P. Lees-Miller
Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends
Nucleic Acids Res., August 16, 2004; 32(14): 4351 - 4357.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
C. Beger, M. Ramadani, S. Meyer, G. Leder, M. Kruger, K. Welte, F. Gansauge, and H. G. Beger
Down-Regulation of BRCA1 in Chronic Pancreatitis and Sporadic Pancreatic Adenocarcinoma
Clin. Cancer Res., June 1, 2004; 10(11): 3780 - 3787.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. Zhang, H. Willers, Z. Feng, J. C. Ghosh, S. Kim, D. T. Weaver, J. H. Chung, S. N. Powell, and F. Xia
Chk2 Phosphorylation of BRCA1 Regulates DNA Double-Strand Break Repair
Mol. Cell. Biol., January 15, 2004; 24(2): 708 - 718.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. M. Y. Cheung, M. P. Hande, F. Jalali, M.-S. Tsao, B. Skinnider, A. Hirao, J. P. McPherson, J. Karaskova, A. Suzuki, A. Wakeham, et al.
Loss of Brca2 and p53 Synergistically Promotes Genomic Instability and Deregulation of T-cell Apoptosis
Cancer Res., November 1, 2002; 62(21): 6194 - 6204.
[Abstract] [Full Text] [PDF]