Impact of RAD51C-mediated homologous recombination on genomic integrity in Barrett’s adenocarcinoma cells

Jagannath Pal, Purushothama Nanjappa, Subodh Kumar, Jialan Shi, Leutz Buon, Nikhil C. Munshi, Masood A. Shammas

Abstract


Background: In normal cells, RAD51-mediated homologous recombination (HR) is a precise DNA repair mechanism which plays a key role in the maintenance of genomic integrity and stability. However, elevated (dysregulated) RAD51 is implicated in genomic instability and is a potential target for treatment of certain cancers, including Barrett’s adenocarcinoma (BAC). In this study, we investigated genomic impact and translational significance of moderate vs. strong suppression of RAD51 in BAC cells.

Methods: BAC cells (FLO-1 and OE33) were transduced with non-targeting control (CS) or RAD51-specific shRNAs, mediating a moderate (40-50%) suppression or strong (80-near 100%) suppression of the gene. DNA breaks, spontaneous or following exposure to DNA damaging agent, were examined by comet assay and 53BP1 staining. Genomewide expression was monitored by microarrays (Affymetrix). Genomic instability was evaluated by Alu-PCR and Homologous recombination (HR) and single strand annealing (SSA) activities measured using plasmid based assays.

Results: We previously showed that suppression of RAD51 significantly reduces the acquisition of genomic changes over time, as assessed by SNP arrays (Affymetrix). Here, we show that although moderate suppression consistenly inhibits/reduces HR activity, the strong suppression is associated with increase in HR activity (by ~15 - ³ 50% in various experiments), suggesting activation of RAD51-independent pathway. Contrary to moderate suppression, a strong suppression of RAD51 is associated with a significant increase in spontaneous as well as UV-induced DNA breaks as well as altered expression of genes involved in detection/processing of DNA breaks and apoptosis. Stronger RAD51 suppression was also associated with mutagenic single strand annealing mediated HR with evidence of increased genomic instability. Suppression of RAD51C inhibited RAD51-independent (SSA-mediated) HR, indicating its role in this process.

Conclusion: Elevated (dysregulated) RAD51 in BAC is implicated in both the repair of DNA breaks as well as ongoing genomic rearrangements. Moderate suppression of this gene reduces HR activity, whereas strong or near complete suppression of this gene activates RAD51C-dependent HR involving a mechanism known as single strand annealing (SSA). SSA-mediated HR, which is a mutagenic HR pathway, further disrupts genomic integrity by increasing DNA breaks and acquisition of new genomic changes in BAC cells. We, therefore, conclude that inhibition of RAD51C, alone or in combination with RAD51 suppression, has potential to make BAC cells static.


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