TY - JOUR
T1 - Radiosensitivity is predicted by DNA end-binding complex density, but not by nuclear levels of band components
AU - Ismail, Sheikh M.
AU - Buchholz, Thomas A.
AU - Story, Michael
AU - Brock, William A.
AU - Stevens, Craig W.
N1 - Funding Information:
The authors would like to thank Dr Jinsong Liu for providing the homozygous BRCA −/− cell line and Ms. Cora Bartholomew for her assistance with the preparation of this manuscript. This work was supported by grant numbers CNE-98699 from the American Cancer Society (C.W.S.), BC980154, a USAMRMC Breast Cancer Research Program Career Development Award (T.A.B.), and CA06294 (M.S.).
PY - 2004/9
Y1 - 2004/9
N2 - We previously determined that the density of a rapidly migrating DNA end-binding complex (termed 'band-A') predicts radiosensitivity of human normal and tumor cells. The goal of this study was first to identify the protein components of band-A and to determine if the protein levels of band-A components would correlate with band-A density and radiosensitivity. DNA end-binding protein complex (DNA-EBC) protein components were identified by adding antibodies specific for a variety of DNA repair-associated proteins to the DNA-EBC reaction and then noting which antibodies super-shifted various DNA-EBC bands. Band-A levels were correlated with SF2 for a panel of primary human fibroblasts heterozygous for sequence-proven mutations in BRCA1 or BRCA2. The nuclear protein levels of band-A components were determined in each BRCA1 heterozygote by western hybridization. DNA-EBC analysis of human nuclear proteins revealed 10 identifiable bands. The density of the most rapidly migrating DNA-EBC band correlated closely with both BRCA-mutation status and radiosensitivity (r 2=0.85). This band was absent in cells with homozygous mutations in their ataxia-telangiectasia-mutated protein (ATM) genes. This band was also completely supershifted by the addition of antibodies to ATM, Ku70, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53. However, the intranuclear concentrations of these proteins did not correlate with either the SF2 or DNA-EBC density. Neither BRCA1 or BRCA2 could be detected in band-A. DNA-EBC analysis of human nuclear extracts resulted in 10 bands, at least six of which contained ATM. The density of one of the DNA-EBCs predicted the radiosensitization caused by BRCA haploinsufficiency, and this band contains Ku70, ATM, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53 but not BRCA 1 or 2. The density of band-A was independent of the nuclear concentration of any of its known component.
AB - We previously determined that the density of a rapidly migrating DNA end-binding complex (termed 'band-A') predicts radiosensitivity of human normal and tumor cells. The goal of this study was first to identify the protein components of band-A and to determine if the protein levels of band-A components would correlate with band-A density and radiosensitivity. DNA end-binding protein complex (DNA-EBC) protein components were identified by adding antibodies specific for a variety of DNA repair-associated proteins to the DNA-EBC reaction and then noting which antibodies super-shifted various DNA-EBC bands. Band-A levels were correlated with SF2 for a panel of primary human fibroblasts heterozygous for sequence-proven mutations in BRCA1 or BRCA2. The nuclear protein levels of band-A components were determined in each BRCA1 heterozygote by western hybridization. DNA-EBC analysis of human nuclear proteins revealed 10 identifiable bands. The density of the most rapidly migrating DNA-EBC band correlated closely with both BRCA-mutation status and radiosensitivity (r 2=0.85). This band was absent in cells with homozygous mutations in their ataxia-telangiectasia-mutated protein (ATM) genes. This band was also completely supershifted by the addition of antibodies to ATM, Ku70, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53. However, the intranuclear concentrations of these proteins did not correlate with either the SF2 or DNA-EBC density. Neither BRCA1 or BRCA2 could be detected in band-A. DNA-EBC analysis of human nuclear extracts resulted in 10 bands, at least six of which contained ATM. The density of one of the DNA-EBCs predicted the radiosensitization caused by BRCA haploinsufficiency, and this band contains Ku70, ATM, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53 but not BRCA 1 or 2. The density of band-A was independent of the nuclear concentration of any of its known component.
KW - Ataxia-telangiectasia-mutated protein
KW - BRCA1
KW - Prediction
KW - Radiation
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U2 - 10.1016/j.radonc.2004.07.003
DO - 10.1016/j.radonc.2004.07.003
M3 - Article
C2 - 15450732
AN - SCOPUS:4644267082
SN - 0167-8140
VL - 72
SP - 325
EP - 332
JO - Radiotherapy and Oncology
JF - Radiotherapy and Oncology
IS - 3
ER -