Suganto Sutjipto

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo

Alterations in the p53 tumor‐suppressor gene occur in 35–60% of human glioblastomas, and re‐introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication‐deficient recombinant adenoviruses encoding wild‐type p53 (rAd‐p53). Western blot analyses showed high‐level expression of p53 protein after treatment with rAd‐p53, and transgene expression was dependent on promoter strength. A p53‐specific dose‐dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus‐mediated gene transfer and expression. p53‐specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd‐p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd‐p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re‐introduction of wild‐type p53 may have potential clinical utility for gene therapy of glioblastoma.

Empty Capsids in Column-Purified Recombinant Adenovirus Preparations

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

Intratumoral Spread and Increased Efficacy of a p53-VP22 Fusion Protein Expressed by a Recombinant Adenovirus

ABSTRACT In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.