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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 29, 2001 as doi:10.1096/fj.01-0500fje. |
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Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany
2Correspondence: Max Delbrück Center for Molecular Medicine, Wiltbergstr. 50, D-13125 Berlin, Germany. E-mail: cardoso{at}fvk-berlin.de
SPECIFIC AIM
Transgenic expression of SV40 large T antigen in terminally differentiated cells can overcome their proliferative block, but it leads to genetic modification of target cells and constitutes a potential hazard. To circumvent these problems, we have taken advantage of the intercellular trafficking properties of VP22, a herpes simplex virus I tegument protein, to test 1) whether it can directly cargo SV40 TAg protein into terminally differentiated cells and 2) whether the transduced VP22-TAg chimeric protein is able to induce a proliferative response.
PRINCIPAL FINDINGS
1. VP22 mediates the transfer of SV40 large T antigen into the nuclei of terminally differentiated skeletal muscle cells
To test whether HSV-1 VP22 can transport SV40 TAg into terminally differentiated cells, we constructed a translational fusion in which the SV40 TAg containing a carboxyl-terminal his/myc tag was fused in frame at the carboxyl terminus of HSV-1 VP22. As a model system for differentiation, we chose the well-characterized skeletal muscle system. Skeletal myoblasts proliferate in culture and can be induced to differentiate under mitogen deprivation conditions. This differentiation process can be unequivocally monitored as myoblasts fuse and form characteristic multinucleated myotubes that express muscle-specific proteins and are permanently withdrawn from the cell cycle. To analyze the trafficking ability of the VP22-TAg chimeric protein into skeletal muscle cells, we performed coculture experiments of transfected COS-7 cells (producing cells) with terminally differentiated C2C12 myotubes (recipient cells). Cocultures with mock transfected COS-7 cells were performed as a negative control. After 24, 48, and 72 h, the mixed cultures were fixed with methanol or formaldehyde and immunostained for his tag or SV40 large T antigen to detect the presence of transduced VP22-TAg protein in myotubes. Myotubes that were cocultured with VP22 large T antigen-expressing COS-7 cells accumulated the chimeric fusion protein in the nuclei. These data clearly indicate that large T antigen can be transferred into the myotube nuclei using VP22 as a vehicle.
2. VP22 large T antigen induces cell cycle reentry in terminally differentiated muscle cells
To address the question of whether the delivered large T antigen is still active and able to induce cell cycle reentry even in fusion with VP22, we assayed myotubes cocultured for 24, 48, or 72 h with VP22 large T antigen-expressing COS-7 cells for incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA and induction of proliferating cell nuclear antigen (PCNA), which is necessary for DNA replication and not expressed in differentiated myotubes. Using antibodies raised against SV40 large T antigen (mouse monoclonal antibody), BrdU (rat monoclonal antibody), and PCNA (rabbit polyclonal antibody) we performed indirect immunofluorescence stainings of the cocultures, which had been incubated with BrdU for 24 h before formaldehyde fixation. Microscopical analysis revealed that the VP22-TAg was indeed able to induce S-phase reentry, as seen by the positive BrdU signals and induction of the replication factor PCNA. Figure 1
shows an example of a myotube that accumulated VP22 large T antigen in its nuclei. This direct delivery of the chimeric protein resulted in S-phase reentry as indicated by induction of PCNA and incorporation of BrdU into the replicating DNA. In contrast, no BrdU or PCNA positive nuclei were detected in the control myotubes cocultured with mock transfected COS-7 cells. VP22 large T antigen was also able to induce mitosis in terminally differentiated muscle cells.
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CONCLUSIONS AND SIGNIFICANCE
The goal of this study was to explore a novel approach to reverse terminal differentiation. We took advantage of the ability of HSV-1 VP22 to transduce fused proteins into cells and tested whether direct protein delivery of SV40 large T antigen via fusion to VP22 could induce cell cycle reentry in terminally differentiated muscle cells. We show here that VP22 can cargo SV40 large T antigen into terminally differentiated skeletal muscle cells, which as a result reenter the mitotic cell cycle (Fig. 2
).
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In addition to viral oncogenes, another attractive candidate is the msx1 transcriptional repressor, which was recently shown to induce C2C12 myotube dedifferentiation. Future experiments will show whether other combinations of regulatory factors, including msx1, can be delivered with this approach and elicit a regenerative response. This protein delivery strategy could easily be combined with application of other compounds such as, e.g., myoseverin, a newly reported small microtubule binding purine shown to induce myotube cytokinesis.
This approach also offers new possibilities for investigating the effects of other factors in terminally differentiated skeletal muscle cells. Since these cells are quite resistant to most gene transfer methods, direct protein delivery provides a novel alternative to apply regulatory factors in a time- and dose-controlled manner to living cells.
Finally, VP22-derived particles designated Vectosomes have recently been shown to cargo proteins as well as nucleic acids into cells, where they remain stable until they are released by light stimulation. This light-induced release of cargo could be used for a temporally and spatially controlled delivery of therapeutic factors.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0500fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0500fje 
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