Abstract 1757: The effect of Zc3h8 expression levels on sensitivity to DNA damage and repair in mouse mammary cells

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Abstract Localized in PML nuclear bodies, the fetal liver zinc finger protein (Fliz), or ZC3H8, has been shown to increase cell proliferation, migration, and tumorigenesis in mouse mammary cell lines. While the exact function of ZC3H8 is unclear, PML nuclear bodies are critical centers for genome maintenance. Previous studies have suggested that these nuclear bodies act as deposits of sequestered DNA damage response proteins including SUMO-1, TP53, and BRCA-1. In addition to their localization to PML nuclear bodies, the phosphorylation of ZC3H8 by CK2 leads to dispersion of PML nuclear foci. For these reasons, we speculated that increased expression of Zc3h8, leading to greater protein availability, is involved in the suppression of DNA repair. This in return would increase genome instability and cancer cell aggression. This study uses the single cell comet assay to measure DNA damage repair following treatment with the topoisomerase inhibitor and chemotherapy drug etoposide. Two mouse mammary tumor cells lines, cV1A 01-51 and cV1A 03-31 were transfected to drive an shRNA targeting ZC3H8 (Flizout). In addition, the cV1A 03-31 Flizout line was rescued with a Zc3h8 construct with altered base coding to make transcribed products immune to shRNA targeting (SynFliz). We have also developed a constitutively charged cV1A 03-31 T32E mutant and a non-phosphorylatable T32A mutant. The nontumorigenic Comma1-D cell line was transfected with a vector to increase Zc3h8 expression. Ability to repair after etoposide treatment in these mutants was compared to controls carrying endogenous levels of Zc3h8. Etoposide leads to double strand breaks in the DNA during replication, and all cell lines were sensitive to etoposide and able to undergo repair. For the cV1A 01-51 lines, the control and Flizout mutants had a nonsignificant difference in post-repair comet length (p = 0.06) but a significant difference in tail moment (p = 0.003). Between the control and the mutants, the difference in overall comet length was 4.7%, and the difference in tail moment was 15.8%, with cells expressing higher levels of Zc3h8 being less adept in carrying out repair of double strand DNA breaks. This suggests an inverse relationship between Zc3h8 expression and DNA damage repair. This may indicate that evaluating Zc3h8 expression could be considered for determining the ability of tumor cells to respond to chemotherapy agents. Furthermore, altering its expression may be a novel approach for cancer treatment. Citation Format: Spencer Li, John A. Schmidt, Janice E. Knepper. The effect of Zc3h8 expression levels on sensitivity to DNA damage and repair in mouse mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1757.