Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts: implications for vector-dependent plasmid transfection.
The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.
Pellegrin
Pierre
P
Institut de Génétique Humaine, Centre National de la Recherche Scientifique UPR 1142, Montpellier, France.
Fernandez
Anne
A
Lamb
Ned J C
NJ
Bennes
René
R
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005822:Genetic Vectors / Q000378:metabolism*; D006735:Horseradish Peroxidase; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D014162:Transfection
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; C069889:protein kinase inhibitor peptide (6-24); D006735:Horseradish Peroxidase