Adenovirus encoding HIV-1 Vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines.
The targeted delivery of genes whose products arrest the cell cycle and/or induce apoptosis represent an important tool for the understanding and controlling forms of unregulated cell growth. The vpr gene product of HIV-1 has been reported to interfere with cell growth and induce apoptosis, but the mechanism of its action is not clearly understood. In order to study these important properties of Vpr, we created a recombinant adenovirus H5.010CMV-vpr (adCMV-vpr) as a tool to deliver the vpr gene to various cell lines to examine its biology. Vpr protein expression was confirmed by Western blot analysis in adCMV-vpr infected cells. We tested the effects of adCMV-vpr on cell growth of several tumor cell lines. Infection of both p53 positive and p53 deficient tumor cell lines with adCMV-vpr resulted in dramatic induction of cell death in short-term assays. We observed that apoptosis was induced through the mitochondrial pathway as we observed changes in the cytochrome c content accompanied by caspase 9 activation. As Bcl-2 is reported to interfere with apoptosis through the mitochondrial pathway, we examined the effect of adCMV-vpr in Bcl-2 over expressing cell lines. We observed that Bcl-2 overexpression does not inhibit adCMV-vpr induced apoptosis. The properties of adCMV-vpr inducing apoptosis through caspase 9 in a p53 pathway independent manner suggest that this is an important reagent. Such a vector may give insight into approaches designed to limit the growth of pathogenic human cells.
Muthumani
Karuppiah
K
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104, USA.
Zhang
Donghui
D
Hwang
Daniel S
DS
Kudchodkar
Sagar
S
Dayes
Nathanael S
NS
Desai
Brijal M
BM
Malik
Afshan S
AS
Yang
Joo-Sung
JS
Chattergoon
Michael A
MA
Maguire
Henry C
HC
Weiner
David B
DB
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle; D048429:Cell Size; D002470:Cell Survival; D004789:Enzyme Activation; D005434:Flow Cytometry; D017353:Gene Deletion; D015870:Gene Expression; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D016336:Gene Products, vpr; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases