DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated...

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• DNA double-strand breaks (DSBs) are repaired in human cells primarily through the non-homologous end-joining (NHEJ) pathway.
• Deficiencies in NHEJ can lead to error-prone repair of DSBs involving microhomology.
• Accurate NHEJ typically resolves DSBs with incompatible 3' overhangs via overlap/fill-in mechanisms.

Purpose of the Study:

• To investigate the DNA repair mechanisms for DSBs with mismatched ends in human bladder carcinoma tissues.
• To compare the accuracy of DSB repair in bladder tumor extracts versus normal cell lines.
• To determine the pathway responsible for DSB repair in high-grade bladder tumors.

Main Methods:

• Utilized cell-free extracts from human glioblastoma (MO59K) and urothelial (NHU) cell lines for comparison.
• Analyzed DSB repair products from extracts of four high-grade bladder carcinomas.
• Inactivated accurate NHEJ in MO59K cells by inhibiting Ku70 or DNA-PK(cs) to assess alternative pathways.

Main Results:

• Cell-free extracts from normal human cell lines accurately joined DSBs with incompatible 3' overhangs via NHEJ.
• Bladder tumor extracts failed to form accurate joins; instead, they exhibited non-random nucleotide deletions.
• Repair in tumor extracts favored annealing at an 8 nt microhomology, independent of Ku70, DNA-PK, or XRCC4, and persisted even when NHEJ was inhibited.

Conclusions:

• Bladder tumor extracts demonstrate a significant deficiency in accurate NHEJ, leading to predominant error-prone joining.
• High-grade bladder tumors utilize a mutagenic, microhomology-mediated alternative end-joining pathway for mismatched DSBs.
• This alternative repair pathway may significantly contribute to the genomic instability observed in bladder cancer.