Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Abstract

Cultured primary human cells have been widely used to assess the selectivity of oncolytic viruses as potential anticancer agents. As culture conditions can potentially have a significant impact on virus replication and ultimately cell killing, we evaluated the effects of dl309, a wild-type adenovirus, and dl01/07, a conditionally replicating adenovirus mutant, on quiescent and proliferating primary mammary epithelial cells. When primary cells were induced into quiescence, both viruses exhibited similar attenuated cell killing. However, cell killing by dl309 was superior to dl01/07 in proliferating primary cells. Analysis of viral effects at the level of entry, E2F activation, DNA replication, and late gene expression indicated that attenuation of dl309 in quiescent cells correlated with decreased expression of viral late genes such as hexon. In contrast, attenuation of dl01/07 in quiescent cells correlated with inefficient induction of E2F activity and inability to undergo efficient DNA replication. In proliferating cells, dl309 replicated efficiently, whereas dl01/07 still showed attenuated replication. In summary, our results indicate the intrinsic preference of wildtype adenoviruses for killing proliferating cells, which is an attractive feature for using adenoviruses as oncolytic agents. These results also highlight the need for the use of appropriate growth conditions for primary cells in vitro to distinguish subtle differences in cell killing among various oncolytic viruses. Cancer Gene Therapy (2005) R eplication-competent adenoviruses are currently being evaluated as novel anticancer agents. A number of modifications in the genome of the adenovirus have been reported to result in viruses that are capable of selectively replicating in and killing cancer cells. 1,2 Based on their ability to selectively kill tumor cells in cell culture and antitumor activity in xenograft tumor models, a few of these engineered viruses are currently being evaluated as oncolytic agents in the clinic. 3 As human adenoviruses do not replicate well in nonhuman cells, it has not been possible to study toxicity related to replication in nontarget tissues in preclinical animal models. In the absence of suitable in vivo models, analysis of selective replication is widely performed by studying these viruses for their effect in vitro on tumor cells lines and primary nontransformed cells. 4 The effect of oncolytic viruses in vitro has been determined at the level of viral DNA replication, late gene expression, induction of cell death, and production of progeny virion. 5 In the in vitro assays, whether cells are rapidly growing or arrested can potentially have a significant effect on virus replication and ultimately cell killing.In the present study, we sought to compare the effect of the wild-type adenovirus and a mutant that was previously reported to replicate preferentially in tumor cells for their effects in proliferating and quiescent normal cells. Our results show that in vitro, replication competent adenoviruses including wild ...

Antitumor Activity of an Oncolytic Adenovirus-Delivered Oncogene Small Interfering RNA

“…6A), S + G 2 -M cycling cells constituted 81% of dl309-treated cells, 73% of Internavec-treated cells, and 58% of ONYX-411-treated cells at 96 hours post-infection. These findings were consistent with previous reports (28,29) of E1A and E4 early adenoviral genes overriding the fundamental control of the host cell cycle, forcing progression into the cell cycle. Furthermore, Internavec treatment, but not ONYX-411, produced significantly elevated accumulation of cells in S + G 2 -M phases as compared with untreated cultures at 72 hours (68 F 8.4% versus 52 F 0.4%, n = 3; P = 0.028) and 96 hours (71 F 7.2% versus 49 F 3.8%, n = 3; P = 0.01; mean F SD).…”

“…The self-limiting disease course of wild-type adenoviral infections in immunocompromised patients is indicative of limited virulence (29). Host cells with viral protein expression are commonly seen post-infection, indicative of an aborted viral replicative process and/or a latent infection state (13,28,29). Coupled with the common defects in apoptotic and IFN-induction pathways in cancer cells, the aborted oncolytic process is an explanation for incomplete clinical responses seen in oncolytic virotherapy trials (14,18).…”

“…Currently, the precise mechanism by which a conditional replicative adenovirus effects tumor cell kill is unclear. The self-limiting disease course of wild-type adenoviral infections in immunocompromised patients is indicative of limited virulence (29). Host cells with viral protein expression are commonly seen post-infection, indicative of an aborted viral replicative process and/or a latent infection state (13,28,29).…”

Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses

“…We preselected the virus Ad5/3 as optimal for our strategy by screening a virus panel and picking a virus with limited cytopathic effect in MPCs and maximum cytopathic effect in the tumor cell line. It has been shown that wild-type adenoviral replicates and kills highly proliferating cells more efficiently and therefore only exerts an attenuated effect on quiescent cells (48). This could explain the lower cytopathic effect rate of adenoviruses on MPCs compared with tumor cells.…”

Oncolytic viral therapy for human cancer: Challenges revisited

“…Conversely, wild-type adenoviral infections of even immunocompromised patients often produced self-limiting disease, which is indicative of the limited virulence of the adenovirus [Vaillancourt et al, 2005;Hogg, 2001]. The expression of viral proteins by the host cell without replication of a complete virus has been well documented in adenovirus-infected host tissues [Elliott et al, 1995;Rao et al, 2004].…”