Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells.
We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.
Stein
C A
CA
Department of Medicine, Columbia University, New York, New York 10032.
Tonkinson
J L
JL
Zhang
L M
LM
Yakubov
L
L
Gervasoni
J
J
Taub
R
R
Rotenberg
S A
SA
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001693:Biological Transport, Active; D015415:Biomarkers; D002478:Cells, Cultured; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D010873:Pinocytosis / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism
D015415:Biomarkers; C081154:FOdT15; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; C059536:Sd(C)28; D013395:Sucrose; D003562:Cytidine; D011494:Protein Kinases; D011493:Protein Kinase C; D013936:Thymidine