Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.
The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.
Moorman
J P
JP
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Bobak
D A
DA
Hahn
C S
CS
D036002:ADP Ribose Transferases / Q000235:genetics / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001905:Botulinum Toxins; D002459:Cell Fusion / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005822:Genetic Vectors; D016399:Lymphoma, T-Cell / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D020741:rho GTP-Binding Proteins
D017931:DNA Primers; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins