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 [P-32]ADP-ribosylated one protein that was identified as RhoA by immunoblot and two-dimensional gel electrophoresis. [P-32]ADP ribosylation of RhoA in HL60 homogenates in vitro was reduced to 10-20% when cells in culture were pretreated with C3 exoenzyme (10 mu g, 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.