ADP-ribosylation of the GTP-binding protein RhoA blocks cytoplasmic division in human myelomonocytic cells.
To test the role of Rho GTP-binding proteins in growth regulation of human myelomonocytic tumour cells we used recombinant C3 exoenzyme of Clostridium botulinum to specifically ADP-ribosylate and inactivate Rho proteins in situ. In homogenates of HL60 cells, the C3 exoenzyme [32P]ADP-ribosylated one protein that was identified as RhoA by immunoblot and two-dimensional gel electrophoresis. [32P]ADP ribosylation of RhoA in HL60 homogenates in vitro was reduced to 10-20% when cells in culture were pretreated with C3 exoenzyme (10 micrograms, 24 h), indicating that 80-90% of RhoA could be ADP-ribosylated in situ. The C3 exoenzyme inhibited HL60 cell proliferation by up to 80% and the degree of growth inhibition correlated with the amount of in situ ADP-ribosylated RhoA in a time- and dose-dependent manner. Cell cycle analysis demonstrated that the C3 exoenzyme-treated HL60 cells accumulated in mitosis, and nuclear staining revealed binucleated cells. These findings suggest that RhoA has a key role in human myelomonocytic tumour cell growth by regulating cytoplasmic division.
Aepfelbacher
M
M
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, University of Munich, Germany.
Essler
M
M
Luber De Quintana
K
K
Weber
P C
PC
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D015415:Biomarkers; D015153:Blotting, Western; D001905:Botulinum Toxins; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects*; D002467:Cell Nucleus / Q000378:metabolism; D003014:Clostridium botulinum / Q000201:enzymology; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000378:metabolism / Q000494:pharmacology; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein
D015415:Biomarkers; D011994:Recombinant Proteins; D015638:Cytochalasin D; D012266:Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein