Increased but error-prone nonhomologous end joining in immortalized lymphoblastoid cell extracts from adult cancer patients with late radionecrosis.
OBJECTIVE
To study nonhomologous end joining in extracts of two lymphoblastoid cell lines derived from patients with late radionecrosis after radiotherapy. Both cell lines were previously shown to exhibit impaired rejoining of DNA double-strand breaks in a pulse-field gel electrophoresis assay.


METHODS
We used a cell-free system and quantitative real-time polymerase chain reaction, as well as sequencing analysis of end joining products.


RESULTS
Paradoxically, extracts of the two cell lines display increased rates of in vitro end joining of noncohesive termini compared with normal cell extracts. This increase was seen in the absence of added deoxyribonucleoside triphosphates and was sensitive to inhibition by wortmannin. Sequencing of the joined products revealed that, despite increased rates of end joining, the process was error prone with a greater frequency of deletions compared with that observed in normal controls.


CONCLUSIONS
These findings are consistent with the suggestion that a promiscuous, deletion-prone abnormality of nonhomologous end joining might underpin the predisposition of certain radiotherapy patients to late radionecrosis. We hypothesize that some individuals might harbor subclinical defects in nonhomologous end joining that clinically manifest on challenge with high-dose radiation. Because both quantitative and qualitative aspects of end joining have demonstrably been influenced, we recommend that the study of patient samples should involve a combination of quantitative methods (e.g., quantitative real-time polymerase chain reaction), sequencing analysis, and a comparison of multiple join types.
Tan
Wu-Meng
WM
Division of Cellular and Molecular Research, National Cancer Centre, Singapore.
Paterson
Malcolm C
MC
Koo
Ghee Chong
GC
Li
Hui Hua
HH
Price
Allan
A
Loong
Susan L E
SL
D000328:Adult; D002460:Cell Line; D002474:Cell-Free System; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D006801:Humans; D009336:Necrosis / Q000235:genetics / Q000473:pathology; D009369:Neoplasms / Q000473:pathology / Q000532:radiotherapy; D016133:Polymerase Chain Reaction / Q000379:methods; D011832:Radiation Injuries / Q000235:genetics* / Q000473:pathology; D017384:Sequence Deletion / Q000235:genetics*