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.

Ionizing radiation enhances dl922–947-mediated cell death of anaplastic thyroid carcinoma cells

“…Chk1 is phosphorylated as early as 3 hpi, indicating a temporal correlation between gH2AX accumulation and ATR-Chk1 activation. This observation is in agreement with previous studies showing a robust induction of ATR-Chk1 pathway upon infection with wild-type and mutant adenoviruses, including dl922-947 (Connell et al 2011). Moreover, our data suggest a correlation with the replicative stress induced by the virus, as gH2AX is mainly observed in replicative and post-replicative phases.…”

“…gH2AX was mainly observed in replicative and post-replicative phases (S-phase and in the following G2/M and polyploid cells with O4N DNA content) (Fig. 5B), supporting a correlation between gH2AX levels and viral induced replicative stress as reported previously (Connell et al 2011).…”

“…It has been shown that the block of ATR-Chk1 pathways augments dl922-947 cytotoxicity (Connell et al 2011). In this study, we demonstrated that also ATM inhibition could represent an approach to increase dl922-947 cytotoxicity.…”

“…It is known that the cells respond to adenovirus infection by activating a DNA damage response (Lilley et al 2007, Nichols et al 2009, Touchefeu et al 2011; indeed, it has been proposed that the prolonged S-phase induced by the virus could resemble the cellular environment occurring in response to replication-associated DNA damage (Connell et al 2011). Also, the linear adenovirus genome has double-stranded DNA termini that could represent targets for cellular DSB repair pathways.…”

“…Cytotoxicity was evaluated using the sulforhodamine B assay as described previously (Vichai & Kirtikara 2006 (Connell et al 2011). Viral replication in tumor xenografts was evaluated as described previously (Libertini et al 2008).…”