MECHANISM OF UPTAKE OF THE PHOSPHONATE ANALOG S)-1-(3-HYDROXY-2-PHOSPHONYLMETHOXYPROPYL)CYTOSINE (HPMPC) IN VERO CELLS

The cellular uptake of phosphonylmethoxypropyl cytosine (HPMPC) was ch aracterize to gain insight into the molecular properties that allow th is anticytomegalovirus drug to permeate cell membranes. The time cours e of uptake of HPMPC into Vero cells was linear between 10 and 75 min and proportional to the concentration in the medium from 10(-6) to 10( -2) M. HPMPC uptake was temperature sensitive and the rate of uptake w as considerably lower at 27-degrees than at 37-degrees and almost tota lly inhibited at 4-degrees. In competition studies with naturally occu rring nucleosides, nucleotides or the phosphonylmethoxyethyl derivativ es, none affected the uptake of HPMPC at concentrations up to 2000-fol d molar excess. The uptake of [H-3]HPMPC into Vero cells was compared with that of [C-14]sucrose, a probe for fluid-phase endocytosis. Kinet ics for both compounds were very similar, as were the effects of the m icrotubule antagonist colchicine and the tumor promoting agent phorbol myristate acetate. Colchicine and the phorbol ester are known to, res pectively, inhibit and stimulate endocytosis. It is concluded from the se data that HPMPC enters Vero cells by fluid-phase endocytosis and th at once internalized it may accumulate in the lysosome. Protonation of the negative charge on the phosphonyl group in HPMPC may allow its di ffusion across the lysosome membrane and eventual activation to its pu tative active diphosphorylated form in the cell cytoplasm.