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Cultured adherent cells can be electroporated in situ, as they grow on a glass slide coated with electrically conductive, optically transparent indium-tin oxide (ITO). Although the introduction of DNA is a common use, the technique of electroporation in situ is valuable for studying many aspects of signal transduction. This is because, under the appropriate conditions, in situ electroporation can be remarkably nontraumatic, while a large variety of molecules, such as peptides, oligonucleotides, or drugs, are introduced instantly and into essentially 100% of the cells, making this technique especially suitable for kinetic studies of effector activation. Following the introduction of the material, the cells can be either extracted or biochemically analyzed, or their morphology and gene expression can be examined by immunocytochemistry. In this chapter, we describe the introduction of a peptide blocking the Src-homology 2 domain of the adaptor Grb2 to inhibit the activation of the downstream effector Erk1/2 by EGF. The setup includes nonelectroporated, control cells growing side by side with the electroporated ones on the same type of ITO-coated surface. In a modified version, this assembly can be used very effectively for studying intercellular, junctional communication: cells are grown on a glass slide half of which is ITO-coated. An electric pulse is applied in the presence of the fluorescent dye lucifer yellow, causing its penetration into the cells growing on the conductive part of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.

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.

Activation of the Ras pathway is central to mitogenesis by a variety of growth factors such as the epidermal growth factor, platelet-derived growth factor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind either directly or indirectly through Src homology 2 domains to the activated receptor. To examine the role of the Src homology 2 domain of growth factor receptor-binding protein 2 in the mitogenic response triggered by these growth factors, we introduced a peptide (PVPE-phosphono-methylphenylalanine-INQS) that can selectively bind this domain into mouse, rat, or human cells growing on conductive indium-tin oxide-coated glass by in situ electroporation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinase (ERK) 1/2, by probing with antibodies specific for its activated form. Electrodes and slides were configured to provide nonelectroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. The data demonstrate that the peptide can cause a dramatic inhibition of epidermal growth factor or platelet-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vivo, compared with its control phenylalanine-containing counterpart. In contrast, the peptide had a very limited effect on hepatocyte growth factor-triggered ERK1/2 activation and DNA synthesis. These results demonstrate the potential of the in situ electroporation approach described here in the study of the coupling of activated receptor tyrosine kinases to the ERK1/2 cascade.

Animal tumor models have demonstrated a close correlation between gap junctional, intercellular communication (GJIC) and tumor metastasis. To examine GJIC levels in human lung carcinoma cells, a novel technique was developed: cells were grown on a glass slide, half of which was coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse which opens transient pores on the plasma membrane was applied in the presence of the fluorescent dye, Lucifer yellow, causing the dye's penetration into the cells growing on the conductive part of the slide. The migration of the dye through gap junctions to the non-electroporated cells growing on the non-conductive area was then observed microscopically under fluorescence illumination. The results show that this is a rapid, precise and highly reproducible assay for GJIC assessment in lines established from lung carcinomas or freshly explanted lung tumor cells. Out of 17 established lines only two had extensive junctional communication, while out of 16 fresh tumor specimens none displayed GJIC. On the other hand, fibroblasts isolated from the same tumors had extensive junctional permeability. The examination of GJIC in a large number of samples could establish a correlation between GJIC and metastasis which might have prognostic value.

The interruption of signaling cascades in intact cells through the introduction of nonpermeant compounds inferred by in vitro studies to specifically inhibit epidermal growth factor (EGF) receptor (EGF-R) function is described. Two nonpermeant [(alkylamino)methyl]acrylophenone derivatives, [(dimethylamino)methyl] acrylo-para-[(benzoylsulfonyl)-oxy]phenone and [(dimethylamino)-methyl]acrylo-para-[(hydroxy-benzoylsulfonyl++ +)-oxy]phenone, were introduced by in situ electroporation into mouse or rat fibroblasts growing on indium-tin oxide-coated glass. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, the extracellular signal-regulated kinase (ERK1/2), by probing with specific antibodies. Electrodes and slides were configured to provide non-electroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. Using this set-up, these compounds could inhibit EGF- but not platelet-derived growth factor (PDGF)-mediated ERK1/2 activation in vivo. These results demonstrate the potential of the in situ electroporation approach for the study of tyrosine kinase action using selective but nonpermeant inhibitors that would otherwise be ineffective in intact cells.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiative insulin signaling pathway, we introduced a leu61-activated ras gene into a Ras-transformable, C3H10T1/2-derived preadipocytic cell line. The results demonstrate that rasleu61 expression in this line blocks differentiation and that this block appears at lower levels than required for full neoplastic transformation. In addition, to examine whether the inability of Rasleu61 to induce differentiation by replacing the insulin signal could be attributed to its transforming effect in this system, we examined the effect of Rasleu61 at levels below the baseline, by expressing rasleu61 in a series of preadipocytes which were rendered deficient in endogenous c-Ras activity. The results show that even very low Rasleu61 levels, insufficient to restore the growth rate of these cells to normal, blocked rather than enhanced differentiation, indicating that rasleu61 expression alone is not sufficient to promote adipocytic differentiation in this system, even in the absence of neoplastic transformation. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the v-Ras effects upon adipocytic differentiation and transformation.

To investigate the role of the cellular ras gene product in neoplastic transformation by the SV40 large tumor antigen (SVLT), murine C3H10T1/2 cells were rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs or through the introduction of the dominant-negative mutant, ras(asn17). Consistent with previous results, SVLT-induced morphological transformation was unaffected by the down-regulation of c-ras gene product activity. On the other hand, colony formation in soft agar and tumorigenicity in nude mice were drastically reduced in c-Ras-deficient cells. In addition, SVLT expression in C3H10T1/2 cells led to increased c-Ras activity, as determined by an increase in the Ras-bound GTP/GTP + GDP ratio. These results suggest that c-Ras is required for full neoplastic transformation by SVLT.

One of the effects of transformation by a variety of factors is a decrease in gap junctional, intercellular communication (GJIC). To investigate the role of the Ras oncogene product in gap junction closure, and to incorporate GJIC into the gamut of transformation-related properties which can be regulated by increasing levels of oncogene expression, a panel of murine C3H10T1/2 and 3T3L1 fibroblasts was constructed in which graded increments of Ras(leu61) could reliably be obtained. The inducibly activated Ras(leu61) protein substantially reduced or eliminated GJIC at levels much lower than those needed for neoplastic transformation, as indicated by acquisition of the ability to proliferate in the absence of anchorage. These results indicate that disruption of GJIC, although necessary, is not sufficient for neoplastic transformation in this system.

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.

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.

Endogenous, cellular Ras proteins (c-Ras) mediate the transforming action of the polyoma virus middle Tumor antigen (mT), which is accompanied by elimination of gap junctional, intercellular communication (GJIC). In this report we show that reducing the c-Ras content of murine C3H10T1/2 fibroblasts (10T1/2) through the expression of an anti-sense ras gene, increased GJIC by 60-80% mT totally eliminated GJIC in normal 10T1/2 cells but it reduced GJIC no more than 50% in the c-Ras deficient lines. These results indicate that endogenous c-Ras is at least partly responsible for the mT-induced gap junction closure.

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells by in situ electroporation are described. Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. The migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

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.