Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.
We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells.
Tonkinson
J L
JL
Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Stein
C A
CA
D002460:Cell Line; D005089:Exocytosis; D005434:Flow Cytometry; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010755:Organophosphates / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013873:Thionucleotides / Q000378:metabolism*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D010755:Organophosphates; D013873:Thionucleotides; D019793:Fluorescein