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Abstract: SA-PO097

Proteomic Studies of Cisplatinated DNA Binding Proteins Identifies RtcB, a Novel RNA Ligase, as an Essential Regulator of Epithelial Cell Death and DNA Damage Response

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Jayne, Laura A., The Ohio State University, Columbus, Ohio, United States
  • Kim, Ji Young, The Ohio State University, Columbus, Ohio, United States
  • Bai, Yuntao, The Ohio State University, Columbus, Ohio, United States
  • Pabla, Navjotsingh P., The Ohio State University, Columbus, Ohio, United States
Background

Cisplatin is one of the most widely used and effective anti-cancer drugs. The therapeutic efficacy and side-effects of cisplatin are largely dependent on its ability to cause DNA damage in both normal and cancer cells. However, the molecular mechanisms involved in cisplatin-mediated DNA damage response and repair remains incompletely understood. In order to uncover novel proteins involved in the cisplatin-associated DNA damage response, we carried out immunoprecipitation of cisplatin-DNA adducts followed by mass spectrometric analysis of associated proteins. These studies revealed that RNA ligase RtcB is associated with cisplatinated DNA under in vitro and in vivo conditions.

Methods

In order to identify novel proteins associated with cisplatin-mediated DNA damage response, we used a cisplatin-DNA adduct antibody to pull down chromatin-associated proteins in murine kidneys after cisplatin treatment. Mass spectrometric analysis was then carried out to identify the proteins associated with cisplatinated-DNA. In vivo and in vitro siRNA approaches were then used to decipher the functional relevance of identified proteins in cisplatin-mediated renal epithelial cell death and DNA damage signaling. Western blot and immunofluorescence experiments were carried out in epithelial cells and renal tissues to measure the extent of DNA damage as well as the activation of DNA damage response.

Results

Initial pulldown studies followed by mass spectrometric analysis identified RtcB as a previously unknown sensor of cisplatin-associated DNA damage. Functional studies in murine renal epithelial cels showed that RtcB knockdown impairs DNA repair, increases DNA damage response and sensitizes cells to cisplatin-mediated cell death. In vivo siRNA mediated RtcB knockdown resulted in augmented AKI and higher renal epithelial cell death. While RtcB is known to play a role in tRNA splicing and unfolded protein response, our work has identified RtcB as a novel player involved in the DNA damage response and repair.

Conclusion

Together, these results suggest an important role for RtcB in the therapeutic efficacy and toxicities associated with the anti-cancer drug cisplatin.

Funding

  • Other NIH Support