BACKGROUND The possibility was investigated that complex homeostatic mechanisms which maintain polyamine pools in prostate‐derived tumors may differ from those which are typically seen in other tissues and tumors. METHODS Growth sensitivity and various regulatory responses were investigated in three human prostate carcinoma cell lines (LNCaP, DU145, and PC‐3) treated with the inhibitor of S‐adenosylmethionine decarboxylase CGP‐48664 or the polyamine analog N1,N11‐diethylnorspermine (DENSPM), both of which are currently undergoing phase I clinical trial. RESULTS Prostate tumor cell lines were all similarly growth‐inhibited by the inhibitor CGP‐48664 (IC50 values, 1–5 μM at 72 hr), but varied considerably in their sensitivity to DENSPM. The rank‐order for cell‐line growth inhibition by the analog was DU145 > PC‐3 > LNCaP, with IC50 values of 1, 30, and 1,000 μM, respectively. Both compounds depleted intracellular polyamine pools to levels which seemed sufficient to account for inhibition of cell growth. While polyamine enzyme regulatory responses to both CGP‐48664 and DENSPM were typical of those seen in other cell types, regulation of polyamine transport differed distinctly. Based on Vmax determinations, LNCaP cells failed to upregulate transport in response to CGP‐48664, while PC‐3 and LNCaP cells failed to downregulate transport in response to DENSPM. CONCLUSIONS Relative to other cell lines, polyamine transport in prostate carcinoma cell lines was found to be uniquely insensitive to regulation by polyamines or analogs. Although this did not seem to correlate with growth sensitivity to polyamine analogs in vitro, it should be therapeutically exploitable in in vivo systems. Prostate 34:51–60, 1998. © 1998 Wiley‐Liss, Inc.

Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 microM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the identified pbps may be associated with that function.