Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.

The possibility of introducing definite amounts of nonpermeant molecules into electropermeabilized living cells has been approached by quantifying the amounts of Lucifer Yellow (LY; a 457-Da highly fluorescent molecule) and Phytolacca americana (Pokeweed) antiviral protein (PAP; a 30,000-Da ribosome-inactivating protein) retained by the cells after closure of the electric-field-induced transient structures of permeation. Without the electropermeabilization, these two molecules enter the cell only in very small amounts by fluid-phase pinocytosis. Under our experimental conditions, using the NIH 3T3 cells, the intracellular LY concentration can reach a value equivalent to the extracellular concentration and can be regulated by controlling the external concentration. We describe the use of LY in a rapid and efficient test for the determination of the best electrical-shock conditions of other cell lines. After electropermeabilization, PAP is 2 X 10(5) times more cytotoxic. Its toxicity can be detected at external concentrations (10(-11) M) corresponding to less than 10 internalized molecules per electropermeabilized cell. Therefore, after electropermeabilization, the nonpermeant molecules have a direct access to the cytosol and the biological effect of nonpermeant substances can be revealed.

[1]  P. Seglen,et al.  Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes , 1984, The Journal of cell biology.

[2]  J. Teissié,et al.  Electric field induced transient pores in phospholipid bilayer vesicles. , 1981, Biochemistry.

[3]  T. Tsong,et al.  Survival of sucrose-loaded erythrocytes in the circulation , 1978, Nature.

[4]  A. Skoultchi,et al.  Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. , 1986, Molecular and cellular biology.

[5]  U. Zimmermann,et al.  Electric field-mediated fusion and related electrical phenomena. , 1982, Biochimica et biophysica acta.

[6]  R. Steinman,et al.  Endocytosis and the recycling of plasma membrane , 1983, The Journal of cell biology.

[7]  P Berg,et al.  Introduction of liposome-encapsulated SV40 DNA into cells. , 1980, The Journal of biological chemistry.

[8]  A. Hayday,et al.  Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells , 1986, Molecular and cellular biology.

[9]  H. Hama-Inaba,et al.  Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants. , 1983, Mutation research.

[10]  P. Seglen,et al.  Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes. , 1987, The Biochemical journal.

[11]  A. Pihl,et al.  Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells. , 1980, Experimental cell research.

[12]  M. Scrutton,et al.  Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electropermeabilised human platelets. , 1986, European journal of biochemistry.

[13]  M. Schaefer-Ridder,et al.  Liposomes as gene carriers: efficient transformation of mouse L cells by thymidine kinase gene. , 1982, Science.

[14]  W. W. Stewart,et al.  Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer , 1978, Cell.

[15]  J. Teissié,et al.  Electric field mediated transformation: isolation and characterization of a TK+ subclone. , 1985, Biochemical and biophysical research communications.

[16]  George,et al.  Surface IgM-kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. , 1968, Cancer research.

[17]  J. L. Biedler,et al.  Cellular resistance to actinomycin D in Chinese hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. , 1970, Cancer research.

[18]  T. Nagata,et al.  Introduction of Functional RNA into Plant Protoplasts by Electroporation , 1986 .

[19]  D. Friend,et al.  Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles , 1983, Cell.

[20]  J. Irvin.  Purification and partial characterization of the antiviral protein from Phytolacca americana which inhibits eukaryotic protein synthesis. , 1975, Archives of biochemistry and biophysics.

[21]  E. Neumann,et al.  Gene transfer into mouse lyoma cells by electroporation in high electric fields. , 1982, The EMBO journal.

[22]  U. Zimmermann,et al.  Enzyme loading of electrically homogeneous human red blood cell ghosts prepared by dielelctric breakdown. , 1976, Biochimica et biophysica acta.

[23]  L. Barbieri,et al.  Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed). , 1982, The Biochemical journal.

[24]  F. Stirpe,et al.  Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. , 1980, The Journal of biological chemistry.

[25]  J. Swanson,et al.  Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages , 1985, The Journal of cell biology.