One of the effects of neoplastic transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). The investigation of junctional permeability is usually conducted through the microinjection of the fluorescent dye, Lucifer yellow, followed by observation of its migration into neighboring cells. This is a time-consuming approach, requiring expensive equipment. To overcome these problems, a novel technique was devised which takes advantage of the ability of short electric pulses to create transient "pores" on the cell membrane through which Lucifer yellow can enter, simultaneously and into large numbers of cells, with minimal disturbance to cellular metabolism. Cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse was applied in the presence of Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area was microscopically observed under fluorescence illumination. Using this technique, we investigated the relationship between expression of the middle tumor antigen of polyoma virus (mT) and GJIC in two representative cell systems with different responses to mT. The results show that low mT expression levels, although unable to transform rat F111 cells fully, are able to interrupt GJIC. Although parts of this mechanism might be mediated through protein kinase C (PKC), mT appears to have additional functions. PKC, however, had the opposite effect upon junctional permeability in a clone of mouse NIH-3T3 fibroblasts; intercellular communication in these cells appears to require PKC activity.

A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.

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The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.The DNA tumor virus simian virus 40 has been associated with human malignancies, particularly malignant mesothelioma (1, 2). Neoplastic transformation by this virus is mediated mainly by its large tumor antigen (SVLT), 1 a nuclear oncoprotein capable of transforming a variety of mammalian cell types (3,4). Deletion mutagenesis studies revealed that this ability may result from its interaction with a number of cellular gene products, including the tumor suppressor proteins, p53 and members of the retinoblastoma susceptibility gene product (pRb) family (pRb, p107, p130; reviewed in Refs. 5 and 6). SVLT binds to pRb family members and inactivates their ability to restrain cell proliferation.We have previously demonstrated that neoplastic transformation by SVLT requires the activity of the cellular Ras protooncogene product (c-Ras or Ras), which is a key player in a pathway that relays signals from membrane tyrosine kinases to the nucleus (7,8). SVLT was unable to fully transform cells where endogenous Ras levels were reduced through the introduction of a Ras antisense construct or the dominant-negative mutant, Ras N17 (9). Later work (10) also showed that pRb inactivation in cells from pRb-null mice causes a dramatic increase in Ras activity. To investigate whether SVLT might be activating c-Raf (Raf), the most prominent Ras downstream target, recently shown to be important in opposing apoptosis (11), we examined the effects of SVLT upon Raf catalytic activity. To examine whether activation of this pathway by SVLT requires extensive protein synthesis, we reconstituted the system in baculovirus/Sf9 insect cells, currently the most widely used model for measuring the activity of putative Raf regulators (12). In conjunction with insect cell systems, we examined these relationships in mammalian cells, where these components were either stably or transiently expressed through transfection. The results show that, aside from Ras, SVLT can also activate Raf. Interestingly, this effect may be, at least in part, Ras-independent since SVLT-mediated Raf activation can take place in cell lines where Ras levels have been reduced through antisense Ras expression. EXPERIMENTAL PROCEDURESCell Culture and Gene Expression-SVLT-expressing rat F111 cells were prepared through...