Gene therapy withE2F-1up-regulates the protein kinase PKR and inhibits growth of leiomyosarcomain vivo

Abstract

Overexpression of the transcription factor E2F-1 induces apoptosis in a variety of carcinoma cells and inactivates murine double minute protein 2, a factor associated with poor prognosis in soft tissue sarcomas. We have shown previously that the double-stranded RNA-activated protein kinase PKR plays an important role in mediating this apoptotic response in carcinoma cells to E2F-1. We sought to evaluate the potential of E2F-1 gene therapy in soft tissue sarcomas and to study the involvement of PKR in the response to E2F-1 overexpression in mesenchymal cells. A replication-deficient adenovirus carrying the E2F-1 gene (Ad5E2F) was used to induce E2F-1 overexpression in the p53 mutated leiomyosarcoma cell line, SKLMS-1. Western blot analysis confirmed E2F-1 overexpression and up-regulation of the antiapoptotic factor Bcl-2 48 hours following infection with Ad5E2F. Apoptosis in Ad5E2F-treated cells was confirmed by fluorescence-activated cell sorting analysis and by poly(ADP-ribose) polymerase cleavage and DNA fragmentation assays. Vector-dependent up-regulation of PKR correlated with the amount of Ad5E2F-induced apoptosis. In vivo treatment of SKLMS-1 tumor-bearing BALB/c mice with intratumoral injections of Ad5E2F at a dose of 2 × 1010 viral particles resulted in significant inhibition in tumor growth compared with control-treated animals (P < 0.016). Complete disappearance of all tumors was seen in two of seven mice in the Ad5E2F-treated animals. Immunohistochemical analysis of tumor specimens showed overexpression of E2F-1 and up-regulation of PKR in Ad5E2F-treated tumors. These findings show that adenovirus-mediated overexpression of E2F-1 results in up-regulation of PKR and significant growth suppression of leiomyosarcomas in vivo. Taken together, these data suggest that E2F-1 gene therapy and PKR modulation might be a promising treatment strategy for these tumors that are highly resistant to conventional therapies.

Adenovirus E2F1 Overexpression Sensitizes LNCaP and PC3 Prostate Tumor Cells to Radiation In Vivo

“…In concordance with previous reports from non-prostate tumor models (13, 14), Ad-E2F1 injection alone reduced TV in orthotopic LNCaP tumors (Tables 1 and 2). …”

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

“…Concurring with these results, E2F-1 overexpression induces PKR expression and autophosphorylation (Fig. 5), leading to phosphorylation of its downstream target eIF-2␣ and to apoptotic cell death in a variety of carcinoma cells (367,369). PKR can interact with STAT1 to regulate its DNA-binding activity (381); PKR also can interact with and activate STAT3 in response to the cell growth regulator factor PDGF (73).…”

Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells

“…Preclinical studies using overexpression of E2F1 have shown the induction of apoptosis in several types of human cancer cells, including glioma, melanoma, pancreatic, colon and non-small-cell lung cancers. 9,[37][38][39][40] In all cases, the adenoviral expression of E2F1 resulted in growth suppression, 41,42 suggesting that modulation of E2F1-induced apoptosis could be a promising therapeutic approach in several types of cancer. It has been shown that E2F1 sensitizes cells to chemotherapeutic agents 43 and that the combination of E2F1 gene therapy and chemotherapy produces a synergistic effect on melanoma cell apoptosis.…”