PATTERNS OF INTRACELLULAR COMPARTMENTALIZATION, TRAFFICKING AND ACIDIFICATION OF 5'-FLUORESCEIN LABELED PHOSPHODIESTER AND PHOSPHOROTHIOATEOLIGODEOXYNUCLEOTIDES IN HL-60 CELLS

We have examined the intracellular compartmentalization and traffickin g of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligo s (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean i ntracellular fluorescence determined by flow cytometry. The F signal w as normalized by the addition of the ionophore monensin. An increase i n signal intensity following addition of monensin indicated that the o ligo was resident in an acidic intracellular environment. F-PS, but no t F-PO oligos were found to reside in an acidic environment. An except ion was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidi fied. 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 w as occuring in endosomes or vacuoles. F-PO and F-PS oligos were efflux ed from HL60 cells from two intracellular compartments. However, appro ximately 60% of internalized F-PO oligo resided in a 'shallow' compart ment that was turned over rapidly (t(1/2) = 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 t(1/2) = 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 HL6 0 cells.