We have studied the signalling of oncogenic Ras proteins in order to more fully understand the transformation process. Many of these studies analyze the effect of overexpression of one specific cellular enzyme, via microinjection and stable transfection of cells. In these studies, we have identified a translational initiation factor that acts as an oncogene and demonstrated its dependence on Ras signal transduction to achieve transformation. This factor was the first component of protein synthesis identified that can play an active role in neoplastic transformation. PLCgamma induced DNA synthesis after microinjection into quiescent NIH 3T3 cells, suggesting a role for PLCgamma during the G1 restriction point. PLCgamma1 mutants with defective lipase activity still induced intermediate levels of DNA synthesis. Recently, we have demonstrated that purified PLCgamma src homology peptides induce DNA synthesis after injection into quiescent NIH 3T3 cells. These results illustrate that in addition to catalytic activity, the structure of a protein also can play an important role in signalling. To further examine the role of PLCgamma during the cell cycle and transformation, PLCgamma was transfected into NIH 3T3 cells and stable transfectants were cloned. The PLCgamma transfectants (PLC/3T3) expressed elevated levels of PLC and behaved like transformed cells. The PLC/3T3 cells formed foci under G418 selection, grew in an anchorage- independent fashion in soft agar, proliferated in low serum media, have altered cell cycle profiles, and induced tumors after injection into nude mice. This data demonstrates the important role of PLCgamma in cellular physiology and supports the conclusions drawn from the microinjection studies where PLCgamma was shown to play a significant role at the G1/S interface. Analysis of the PLC/3T3 cells and of cell lines generated from tumor tissue showed a novel form of PLCgamma. This tumorigenetic form of PLCgamma is actively being characterized.
