Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

Abstract

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus.

Figure 1

Cytopathic effect of ONYX-015 on GFP and HSP72 transfected glioma cells. Photographs (100× magnification) of RT2-GFP and RT2-HSP72 cells 96 hours after ONYX-015 infection at various dosages are represented. A – RT2-GFP cells mock infected with virus. B – RT2-HSP72 cells mock infected with virus. C – RT2-GFP cells infected at MOI of 100. D – RT2-HSP72 cells infected at MOI of 100. E – RT2-GFP cells infected at MOI of 300. F – RT2-HSP72 cells infected at MOI of 300.

Figure 2

Cytostatic effect of ONYX-015 infection on HSP72 transfected 9L cells. 9L-HSP72 cells were transduced with either AD-GFP (□) or ONYX-015 (■) at a multiplicity of infection of 100. Cell numbers were determined over time (X-axis) by direct counting using a Coulter Counter. Values (Y-axis) represent log of cell number as a fraction of the initial cell number plated.

Figure 3

9L Cell viability after ONYX-015 infection. Cell viability (as % viable cells; y-axis) of 9L-GFP (■) and 9L-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points.

Figure 4

RT2 Cell viability after ONYX-015 infection. Cell viability (as % viable cells; y-axis) of RT2-GFP (■) and RT2-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points.

Figure 5

Burst assays. Burst ratio is given as the virus titer at 72 hours compared to 4 hours of various cell lines (x-axis) infected while 50% confluent with ONYX-015 at MOI of 100. Results are representative of a typical experiment of at least three performed. (*) represents statistical significance (p < 0.05) by Student's t-test between 9L-HSP72 and 9L-GFP. (**) represents statistical significance (p < 0.05) by Student's t-test between RT2-HSP72 and RT2-GFP.