Nucleated cells response to protein electroinsertion.
Application of an electrical pulse field at a strength slightly below the value required for electroporation to a suspension of red blood cells in the presence of membrane xenoproteins leads to the insertion of those proteins in the erythrocyte plasma membrane. This observation is extended to nucleated cells. In the presence of glycophorin A, application of such pulses leads to the insertion of 10(4)-10(5) molecules of glycophorin A per cell in CEM-CM3, Hela S3, and bovine CD8+ T cells. Electroinserted glycophorin A is detected by flow cytometry using anti-glycophorin monoclonal antibodies. The survival of the cells subjected to electroinsertion was 55% for CEM-CM3 cells, 69% for Hela S3 cells, and 65% for CD8+ T cells. Cells cultured after electroinsertion lost the electroinserted glycophorin A, with two different rates, by a temperature and cell type-dependent mechanism. During the first 2 h after electroinsertion, the CD8+ T cells lost 12.5% of the inserted glycophorin A per h, the CEM-CM3 cells lost 7.7% per h, whereas the Hela S3 cells lost only 0.8% of the inserted protein per h. After 2 h, the rate increased substantially, to 41.7% per h for the CD8+ T cells, 13.5% for the CEM-CM3 cells, and 8.9% for the Hela S3 cells. Cytochalasin D efficiently inhibited the disappearance of electroinserted glycophorin A during the first 2 h after electroinsertion only.
Mouneimne
Y
Y
Center for Blood Research Laboratories, Boston, Massachusetts.
Brown
W C
WC
Nicolau
C
C
Tosi
P F
PF
D000818:Animals; D002417:Cattle; D015638:Cytochalasin D / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D018274:Electroporation; D005057:Eukaryotic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006021:Glycophorins / Q000008:administration & dosage*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006021:Glycophorins; D015638:Cytochalasin D; D004121:Dimethyl Sulfoxide