Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells
Claire M. Connell, Atsushi Shibata, Laura A. Tookman, Kyra M. Archibald, Magdalena B. Flak, Katrina J. Pirlo, Michelle Lockley, Sally P. Wheatley, Iain A. McNeish
Claire M. Connell, Atsushi Shibata, Laura A. Tookman, Kyra M. Archibald, Magdalena B. Flak, Katrina J. Pirlo, Michelle Lockley, Sally P. Wheatley, Iain A. McNeish
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Research Article Oncology

Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells

Abstract

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

Authors

Claire M. Connell, Atsushi Shibata, Laura A. Tookman, Kyra M. Archibald, Magdalena B. Flak, Katrina J. Pirlo, Michelle Lockley, Sally P. Wheatley, Iain A. McNeish

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Figure 1

Positive correlation between virus sensitivity and overreplication phenotype after dl922-947 infection in ovarian cancer cell lines.

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Positive correlation between virus sensitivity and overreplication pheno...
(A) Cell survival in TOV21G, IGROV1, and A2780CP cells was assessed by MTT assay 120 hpi with dl922-947 and Ad5 WT. (B) Survival of TOV21G, IGROV1, and A2780CP cells 120 hpi with dl922-947 at MOIs that permit 50% cell infectivity (MOI 1, 5, and 2 PFU/cell, respectively; data not shown). ***P < 0.001. (C) Expression of E1A was assessed by quantitative RT-PCR and immunoblot after infection with dl922-947 (MOI 7.5). Transcription is normalized to β-actin and presented relative to that of uninfected cells. Error bars (representing SD) are plotted on all data points but may be too small to be visible. (D) TOV21G, IGROV1, and A2780CP cells were harvested up to 72 hpi with dl922-947 (MOI 7.5), fixed in 70% cold ethanol, stained with PI, and analyzed by flow cytometry. >4N, cells with more than 4 N DNA content; 4N, cells with 4 N DNA content; S, cells in S phase; 2N, cells with 2 N DNA content. (E) Hct116 cells were harvested up to 72 hpi with dl922-947 (MOI 7.5), fixed in 70% cold ethanol, stained with PI, and analyzed by flow cytometry. IC50 for Hct116 cells 120 hpi with dl922-947 is approximately 0.04 PFU/cell (data not shown). (F and G) Replication of dl922-947 was assessed in TOV21G, IGROV1, and A2780CP cells after infection with dl922-947 (MOI 7.5) by quantitative PCR (E) 48 and 72 hpi and (F) by TCID50 72 hpi.