Faculty Bibliography

Oncolytic replicating adenoviruses are a promising new modality for the treatment of cancer. Despite the assumed biologic advantage of continued viral replication and spread from infected to uninfected cancer cells, early clinical trials demonstrate that the efficacy of current vectors is limited. In xenograft tumor models using immune-incompetent mice, wild-type adenovirus is also rarely able to eradicate established tumors. This suggests that innate immune mechanisms may clear the virus or that barriers within the tumor prevent viral spread. The aim of this study was to evaluate the kinetics of viral distribution and spread after intratumoral injection of virus in a human tumor xenograft model. After intratumoral injection of wild-type virus, high levels of titratable virus persisted within the xenograft tumors for at least 8 weeks. Virus distribution within the tumors as determined by immunohistochemistry was patchy, and virus-infected cells appeared to be flanked by tumor necrosis and connective tissue. The close proximity of virus-infected cells to the tumor-supporting structure, which is of murine origin, was clearly demonstrated using a DNA probe that specifically hybridizes to the B1 murine DNA repeat. Importantly, although virus was cleared from the circulation 6 hr after intratumoral injection, after 4 weeks systemic spread of virus was detected. In addition, vessels of infected tumors were surrounded by necrosis and an advancing rim of virus-infected tumor cells, suggesting reinfection of the xenograft tumor through the vasculature. These data suggest that human adenoviral spread within tumor xenografts is impaired by murine tumor-supporting structures. In addition, there is evidence for continued viral replication within the tumor, with subsequent systemic dissemination and reinfection of tumors via the tumor vasculature. Despite the limitations of immune-incompetent models, an understanding of the interactions between the virus and the tumor-bearing host is important in the design of effective therapies

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.

Glucocorticoids inhibit the proliferation of various cell types, but the mechanism of this inhibition remains unclear. We investigated the effect of dexamethasone on non-small cell lung cancer cell growth and cell cycle progression. We showed that dexamethasone suppresses the proliferation of A549 and Calu-1 cells, with accumulation of cells in G1/G0 stage of the cell cycle, as determined by fluorescence-activated cell sorter analysis. Western blot analysis confirmed that this is associated with hypophosphorylation of retinoblastoma protein. Using Western blot analysis and in vitro kinase assays, we found that dexamethasone results in decreased activity of CDK2 and 4, decreased levels of cyclin D, E2F, and Myc, and increased levels of the CDK inhibitor p21(Cip1). In addition, we found that dexamethasone decreases activity of extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK). The kinetics of all these changes indicate that inhibition of the ERK/MAPK pathway precedes the cell cycle effects, suggesting that regulation of this MAPK-signaling pathway may be an alternative mechanism for glucocorticoid-induced cell cycle arrest and growth inhibition