Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells

Several lines of evidence suggest that defective repair of DNA double-strand breaks (DSB) contributes to genomic instability in human cancers, including urothelial carcinoma. In particular, extracts from urothelial cancers have been reported to repair DSBs preferentially by microhomology-mediated end-joining (MMEJ), considered as more error-prone than canonical non-homologous end-joining (NHEJ) predominating in normal urothelial cell extracts. However, it is not clear whether such differences are relevant to intact cells. We therefore transfected plasmids digested with different restriction enzymes to yield incompatible ends (blunt, 5′-protruding or 3′-protruding) into urothelial carcinoma cell lines or normal urothelial cells and characterized the recovered circular plasmids. All cells competently repaired DSBs in a standard cloning vector plasmid, processing 5′- as well as 3′-protruding ends. No significant differences in the extent of processing were detected and the junctions presented short microhomologies indicative of canonical NHEJ. However, dramatic and distinctive differences between normal and cancerous urothelial cells were seen in two different experiments. First, cancer cell lines processed a significantly higher fraction of plasmids cut with a single restriction enzyme that could have been repaired by direct ligation than normal cells. Secondly, for the repair of a large plasmid with incompatible ends containing a large fragment of human genomic DNA, normal cells used almost exclusively MMEJ exploiting a microhomology with the 3′-end of the break, whereas cancer cell lines often processed DNA despite suitable microhomologies. DNA repair of the small or large plasmid was almost abolished by siRNA knockdown of Ku70. These findings strongly suggest that urothelial carcinoma cells lack control mechanisms preventing overprocessing during NHEJ repair. This may account for previous findings that urothelical cancers contain unusually large chromosomal deletions. Moreover, in contrast to prevailing interpretations, our observations suggest that MMEJ, despite its error-proneness, in some instances may act as a failsafe mechanism against overprocessing during NHEJ.