Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells
Claire M. Connell, … , Sally P. Wheatley, Iain A. McNeish
Claire M. Connell, … , Sally P. Wheatley, Iain A. McNeish
Published March 7, 2011
Citation Information: J Clin Invest. 2011;121(4):1283-1297. https://doi.org/10.1172/JCI43976.
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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 8

Cdc25A is a key regulator of adenovirus cytotoxicity.

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Cdc25A is a key regulator of adenovirus cytotoxicity. (A) Cdc25A express...
(A) Cdc25A expression was assessed by immunoblot after dl922-947 infection (MOI 7.5). (B and C) A2780CP cells infected with dl922-947 were treated with UCN-01 (300 nM) or MG132 (10 μM) and harvested 48 hpi. (B) Cdc25A and γH2AX expression 24 hpi and (C) E1A expression up to 96 hpi were assessed by immunoblot. (D) 24 hours after transfection with ATR-specific siRNA or scrambled control (Scr), A2780CP cells were infected with dl922-947 (MOI 7.5). ATR and Cdc25A expression was assessed by immunoblot 72 hpi. (E) A2780CP cells were transfected with pX-HA-Cdc25A or pGFP 24 hours before (–24 hours) or 8 hours after (+8 hours) infection with dl922-947 MOI 100. Cell survival was assessed 96 hpi (left). **P < 0.01, ***P < 0.001. γH2AX expression was also assessed in cells infected with dl922-947 and transfected 8 hours later with pX-HA-Cdc25A (middle). Red bars represent median. ***P < 0.0001; Mann-Whitney test. Expression of HA-tagged Cdc25A was confirmed by Cdc25A immunoblot (right). (F) TOV21G cells were transfected with Cdc25A-specific siRNA or scrambled control, infected with dl922-947 (MOI 7.5 and 15) later and stained for γH2AX expression 16 hours thereafter. Red bars represent median. ***P < 0.0001; Mann-Whitney test. (G) A2780CP cells were transfected with Cdc25A-specific siRNA or scrambled control 24 hours prior to infection with dl922-947 (MOI 7.5). Cells were additionally treated with UCN-01 6 hpi and fixed 24 hpi. γH2AX expression (left) and flow cytometry (right) were performed as before. Red bars represent median. ***P < 0.0001; Mann-Whitney test.