SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/ spermine N 1 -acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.Polyamines are essential for normal cellular functions (1, 2). They bind to intracellular polyanions such as nucleic acids and ATP and modulate their functions (3). Intracellular polyamine content is increased in response to growth stimuli (4) and regulated by biosynthesis and degradation (5). Uptake and export also play important roles in the regulation of cellular polyamine levels (5).In recent years, polyamine transporters have been identified in bacteria, yeast, and protozoa, and their properties have been studied. In Escherichia coli, polyamine uptake is mediated by three systems, the spermidine-preferential uptake system PotABCD (6, 7), the putrescine-specific uptake system Pot-FGHI (8), and PuuP (9). Export of polyamines is mediated by PotE (10), CadB (11), and MdtJI (12) in E. coli. Blt is a polyamine exporter in Bacillus subtilis (13). In Saccharomyces cerevisiae, uptake of polyamines is mediated by DUR3, SAM3, GAP1 (14, 15), and AGP2 (16) on the plasma membrane and UGA4 on vacuolar membranes (17). The four transporters TPO1-4 on the plasma membrane (18 -20) and TPO5 on the post-Golgi secretory vesicles (21) are polyamine exporters in yeast. A plasma membrane polyamine transporter, LmPot1, in protozoan parasite Leishmania major (22) has been described. In these unicellular organisms, polyamine transport involves protein channels.In animal cells, polyamine uptake is mediated, at least...

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1-and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.caveolin-1; NOS2; SLC3A2; gastrointestinal tissue POLYAMINES ARE SMALL ORGANIC compounds that have two or more amino groups and are found in almost all organisms (6, 13). The major polyamines found in cells are spermidine and spermine, and their precursor putrescine. Among these, putrescine and spermidine are essential factors for cell growth (4). Polyamines can bind to anions such as DNA, RNA, and ATP and can thereby regulate their functions (9). The major sources of exogenous polyamines come from diet and luminal bacteria (11). An antibiotic treatment to remove microbial flora activity (7) or polyamine-free diet (15) increased the polyamine-depleting effect of the polyamine biosynthetic enzyme inhibitor difluoromethylornithine (DFMO). These results indicate that transport makes a significant contribution to cellular polyamine levels.We have identified the amino acid transporter SLC3A2 as a polyamine export protein in colon cancer-derived cells (24). SLC3A2 associated with the polyamine catabolic enzyme spermidine/spermine N 1 -acetyltransferase, SAT1, and catalyzed the export of acetylated polyamines by the polyamine/ arginine exchange reaction. As for polyamine uptake, we have reported that polyamine uptake was mediated by caveolaedependent endocytosis in colon cancer-derived cells (16).Caveolae are flask-shaped invaginations of plasma membrane with a diameter of 50 -100 nm. They have been implicated in endocytosis and signal transductions...

Phase diagrams of lipid mixtures can show several different regions of phase coexistence, which include liquid-disordered, liquid-ordered, and gel phases. Some phase regions are small, and some have sharp boundaries. The identity of the phases, their location in composition space, and the nature of the transitions between the phases are important for understanding the behavior of lipid mixtures. High fidelity phase boundary detection requires high compositional resolution, on the order of 2% compositional increments. Sample artifacts, especially the precipitation of crystals of anhydrous cholesterol, can occur at higher cholesterol concentrations unless precautions are taken. Fluorescence resonance energy transfer (FRET) can be used quantitatively to find the phase boundaries and even partition coefficients of the dyes between coexisting phases, but only if data are properly corrected for non-FRET contributions. Self-quenching of the dye fluorescence can be significant, distorting the data at dye concentrations that intuitively might be considered acceptable. Even more simple than FRET experiments, measurements of single-dye fluorescence can be used to find phase boundaries. Both FRET and single-dye fluorescence readily detect the formation of phase domains that are much smaller than the wavelength of light, i.e. "nanoscopic" domains.

Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.

Mutation of the Kirsten-ras (Ki-ras) proto-oncogene occurs frequently in colorectal cancers. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki-ras transformation and colon tumorigenesis in carcinogen-treated animal models by mechanisms yet to be elucidated. Caco-2 cells transfected with an activated Ki-ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki-ras-and DFMO-dependent patterns of gene expression. Microarray results were validated with real-time or semi-quantitative RT-PCR and/or Western blot analysis. Genes upregulated in Caco-2 cells expressing an activated Ki-ras encoded cytoskeletal-, transport-, protease-, and gap junction-associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco-2 cells transfected with an activated Ki-ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki-ras-dependent migration was inhibited by INGA1 antibodies. Other Ki-ras-dependent, but DFMO-independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki-ras-transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki-ras oncogene caused changes in experimental cell migration and cell-cell communication genes and that some of these changes could be reversed by DFMO.