A novel technique for the study of intercellular, junctional communication: electroporation of adherent cells on a partly conductive slide.
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.
Raptis
L H
LH
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Brownell
H L
HL
Firth
K L
KL
Mackenzie
L W
LW
D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D002450:Cell Communication / Q000502:physiology*; D002461:Cell Line, Transformed; D002470:Cell Survival; D002471:Cell Transformation, Neoplastic; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005347:Fibroblasts; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D007204:Indium; D007546:Isoquinolines; D051379:Mice; D011493:Protein Kinase C / Q000502:physiology; D017971:Tin Compounds
D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D007546:Isoquinolines; D017971:Tin Compounds; D007204:Indium; C017475:lucifer yellow; D011493:Protein Kinase C; C045358:stannic oxide