ONYX-015 is a promising new cancer gene therapy vector which exerts its tumoricidal effect by selective replication in cancer cells. Replication selectivity previously was attributed to interactions of E1 gene products with p53. Subsequent reports have shown that ONYX-015 replicates in cells with wild-type p53 as efficiently as in cells with mutant-p53. Although the mechanism of cancer cell selectivity is still under investigation, the various hypotheses consistently involve cell-cycle regulatory proteins and their interactions within apoptosis pathways. HSP72 is a potent regulator of apoptosis . Also, there is evidence that activation of the heat shock response by adenovirus early region genes is necessary for virus replication. Previously it has been shown that heat shock induced HSP72 expression resulted in increased production of adenovirus proteins. Additionally, the E1A adenovirus gene products necessary for virus genome replication have previously been shown to co-localize with HSP72.
To study the role of HSP72 in augmenting ONYX-015 replication, we chose two glioma cell lines with different derivations. 9L is a chemically induced gliosarcoma while RT2 is a virally transformed glioblastoma. Compared to many human tumor cell lines, these cell lines show relative resistance to the cytotoxic affects of ONYX-015. Resistance in part was secondary to low adenovirus transduction efficiency. At the multiplicities of infection utilized in this study, we never achieved 100% cell transduction (table 1). In most cases, when less than 30% of permissive HSP72-transfected cells were transduced, tumor cell growth was rapid and outpaced ONYX-015 virus replication resulting in no discernable cytotoxic or cytostatic effect after 1 week in culture (data not presented). Therefore, replicative adenovirus cytotoxicity is likely a balance between virus transduction efficiency, replication efficiency and cell growth characteristics. HSP72 transfection had no bearing on cell doubling time of either 9L or RT2 cells (data not presented) RT2 cell-doubling time is approximately 8 hours while 9L cell-doubling time is approximately 14 hours. Both cell lines after HSP72 transfection showed equal susceptibility to ONYX-015. Further evidence for the balance between transduction efficiency, replication efficiency and cell doubling time is evident in that ONYX-015 infection resulted in a predominantly cytostatic affect resulting in decreased cell division before evidence of cytotoxicity.
The most dramatic affect of the role HSP72 in promotion of virus replication is evident in figure 1. At 96 hours, the GFP transduced cells showed very little cytotoxic effect of ONYX-015 infection. In contrast, there is a distinct cytopathic effect at the same doses of ONYX-015 infection in the HSP72 transduced cells. These cells were rendered susceptible to ONYX-015 toxicity. The mechanism of cell death is related to increased virus replication. Significantly more ONYX-015 was isolated from HSP72 transduced cells than the GFP-transfected controls.
The E1a gene products of adenovirus are responsible for activation of the HSP72 promoter and may affect HSP72 levels during the cell cycle. Conversely, there is no evidence that HSP72 interacts with adenovirus promoters to stimulate viral transcription. There is evidence of HSP72 interactions with adenovirus structural proteins such as hexon and fiber. Adenovirus assembly is known to be inefficient with only a small percentage of total structural proteins eventually utilized in the production of infectious virus .
In conclusion, our studies demonstrated that HSP72 expression in rodent glioma tumor cells potentiated ONYX-015 replication and oncolysis of tumor cells. Further studies on the role of HSP72 in tumor types with wild-type and mutant p53 tumor suppressor genes is warranted to understand the molecular interactions of this chaperone protein in promoting virus replication. Additionally, addressing the role of HSP72 as an inhibitor of apoptosis in virus replication would further help characterize the interaction of cell-cycle regulatory pathways with virus replication. Furthermore, we intend to evaluate the role of HSP72 associated permissive replication in other animal cell lines to establish animal models for the study of ONYX-015 and similar replication competent adenoviruses.