In situ Electroporation for the Measurement of c-Ras Activation by SVLT

Simian virus 40 large-tumor antigen (SVLT) is a largely nuclear oncogene product (1 ,2 ). Evidence from a number of laboratories has indicated that the interaction between cellular nuclear antioncogenes, such as the retinoblastomasusceptibility gene product family (Rb) and p53, mediates neoplastic transformation by SVLT, as outlined in Chapters 14 and 15. However, cytoplasmic deletion mutants, lacking the nuclear localization signal, can still transform established cell lines with efficiencies similar to that of wt SVLT. The demonstration that SVLT requires the function of the membrane-bound, cellular Ras protooncogene product to elicit neoplastic transformation of mouse or rat fibroblasts (3 ), provided an additional link between the nuclear cell cycle machinery and the membrane-signaling apparatus. Later work further revealed that Rb inactivation per se leads to Ras activation (4 ). This communication describes a novel technique to measure c-Ras activation by SVLT, as well as by other Ras stimulators.