Rationale for the Design of Polyamine Fluoroprobes and Initial Studies

At least four laboratories have independently developed fluorescent polyamine analogs as probes to visualize the internalization mechanism and to assess the subcellu-lar distribution of polyamines. Either the terminal (N1) amino group (29,39) or central (N4) amino groups (25,26,40) of a polyamine backbone has been used for conjugating the fluorophore. It is noteworthy that the size of the fluorophore in the conjugates was substantial relative to the polyamine backbone. Except for the fluorescein-derivatized probe (29), which is negatively charged at physiological pH, the fluorescent moieties added to the polyamine (e.g., BODIPY-FL) (Fig. 1A) (26), N-methylanthranyl (Fig. 1B) (25), or 9-anthracenylmethyl (Fig. 1C) (39) are markedly hydrophobic (Fig. 1). Preference for a more hydrophobic fluorophore was based on the assumption that only the polyamine backbone should be impermeant toward the cell membrane and limiting for translocation.

In all cases, the fluorescent polyamine conjugates were shown to be specifically transported via the mammalian polyamine transport system, as shown by competitive inhibition of probe influx by natural polyamines. Moreover, polyamine transport-deficient Chinese hamster ovary (CHO) cell mutants were completely unable to internalize the derivatives (25,26,40). As a further indication that the derivatives behaved

Polyamine Transport
Fig. 1. Structures of three representative fluorescent polyamines used as probes of polyamine transport. (A) Spd^-BODIPY (26), (B) #-{spermidine-[A4-(3-aminopropyl)]}anthranylamide (25), (C) A/-(4-aminobutyl)-A/-anthracen-9-ylmethylbutane-1,4-diamine (39).

like the parent polyamine, prior treatment with DFMO or cycloheximide (a treatment that pre-empts AZ induction and feedback transport inhibition by accumulating substrate) increased the velocity of probe transport (26). Thus the novel fluorescent polyamines physiologically behaved like the parental molecules and required a functional polyamine transport system for their internalization.

Quite remarkably, a feature that was uniformly reported for all fluoroprobes studied was an exclusively cytoplasmic localization of the internalized molecules, as observed by epifluorescence microscopy or by confocal laser scanning microscopy (25,26,29,40). Only extremely weak labeling of chromatin regions could be detected under any circumstances, even in mitotic cells (25). However, the most unexpected finding was the observation that fluoroprobe accumulation was largely confined to vesicle-like structures (25,26,40). The remarkable pattern of these polyamine-sequestering vesicles (PSVs) first led to the suggestion that the polyamine transport process was mediated by receptor-mediated endocytosis rather than classic translocation from the exo- to the endofacial sides of the plasma membrane (25,26). A somewhat similar endocytic mode of polyamine uptake has been proposed to account for the role of glypican-bound heparan sulfate in spermine uptake (41). In the latter model, spermine tightly com-plexed with heparin sulfate chains would be first internalized via endocytic caveolation of glycosylphosphatidylinositol-bound glypican-1 and then be released from the specialized endosomes according to a complex processing of heparin chains involving nitric oxide (41).

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