1-2-dielaidoylphosphatidylethanolamine and Neuroblastoma

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.

Topics: Active Transport, Cell Nucleus; Carcinoma, Hepatocellular; Cell Nucleus; Chromatography, High Pressure Liquid; Circular Dichroism; Cytoplasm; DNA; Dose-Response Relationship, Drug; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Green Fluorescent Proteins; Humans; Intracellular Membranes; Kinetics; Light; Luciferases; Luminescent Proteins; Lysine; Microscopy, Electron; Mitosis; Nanotechnology; Neuroblastoma; Nuclear Pore; Peptides; Phosphatidylethanolamines; Plasmids; Scattering, Radiation; Time Factors; Transfection; Transgenes; Tumor Cells, Cultured

Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (gamma j). Despite a 10-fold variation in gamma j, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gamma j (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2',7'-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na+ and K+ channels), implying similar mechanisms for selectivity.

Topics: Animals; Blotting, Northern; Cells, Cultured; Chickens; Connexins; Diffusion; Electric Conductivity; Electrophysiology; Fluoresceins; Fluorescent Dyes; Gap Junctions; Genetic Vectors; Humans; Ion Channels; Liposomes; Mice; Models, Anatomic; Models, Biological; Molecular Weight; Neuroblastoma; Patch-Clamp Techniques; Phosphatidylethanolamines; Plasmids; Rats; Second Messenger Systems; Surface Properties; Transfection

The Myc family proteins represented by c-Myc are thought to play a crucial role in cellular proliferation, differentiation, transformation, and apoptosis. In this study, we demonstrated the novel role for a Myc family protein in elicitation of immunogenic phenotypes in tumor cells. Injection of rat 9L or C6 glioma cells, together with the s-myc gene linked to the cytomegalovirus promoter, completely prevented formation of both brain tumors and s.c. tumors derived from the parental glioma cells. However, introduction of the s-myc gene had no inhibitory effect on development of B104-derived neuroblastoma. In addition, unlike the s-myc gene, injection of the c-myc or wild type p53 (wt-p53) gene together with glioma cells did not modulate the tumor immunogenicity and resulted in formation of gliomas in the animals. These findings suggest that s-Myc expression may stimulate the presentation of a tumor antigen common to 9L and C6 cells to T lymphocytes and augment the activity of the host immune system, resulting in prevention of glioma formation in vivo. This success in tumor eradication indicates the possibility of application of the s-myc gene for gene therapy of human brain tumors.

Topics: Animals; Brain Neoplasms; Genes, myc; Genes, p53; Genetic Therapy; Glioma; Hindlimb; Male; Neoplasm Transplantation; Neuroblastoma; Phosphatidylethanolamines; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred F344; Skin Neoplasms; Transfection; Tumor Cells, Cultured