Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo.

BACKGROUND: The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. METHODS: LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. RESULTS: In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. CONCLUSIONS: GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.

Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells.

BACKGROUND: Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel. METHODS: Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue. RESULTS: CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI(50)) 3 nM in PANC-1, 5.1 nM in MIA PaCa-2) in combination with 1 µM CYC3 (GI(50) 1.1 µM in MIA PaCa2 and 2 µM in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nM). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nM; 60-70% vs 100% inhibition). CONCLUSION: The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.

Arthroscopic management of the throwing athlete's shoulder: indications, techniques, and results.

This article briefly describes the various pathologic lesions seen in the throwing athlete's shoulder. The pathologic conditions discussed include primary and secondary impingement, tensile lesions of the rotator cuff and biceps-labral complex, glenohumeral laxity, labral tears, and AC joint injuries. Mechanism of injury, indications, and arthroscopic management of these lesions are discussed.

Histamine blocks interleukin 2 (IL-2) gene expression and regulates IL-2 receptor expression.

Histamine inhibited the proliferative response of human peripheral blood mononuclear cells (PBMC) to the T cell mitogen Phytohemagglutinin-P (PHA-P) in a dose-dependent fashion. This inhibition was mediated via the H2 receptor since cimetidine, a known H2 antagonist, removed the inhibition, whereas the addition of the H1 antagonist Diphenhydramine did not. Inhibition occurred during the inductive phase of the cell cycle, since histamine added 24 hours after PHA-P stimulation had no effect on subsequent T cell proliferation, and was attributable to inhibition of interleukin-2 (IL-2) gene expression. Both secreted IL-2 and messenger RNA coding for IL-2 were inhibited by histamine. In contrast, histamine exerted no inhibitory effect on the expression of cell surface receptors for IL-2 as determined by flow cytometry. Furthermore, histamine-treated cells retained full responsiveness to exogenously administered IL-2, which completely reversed the anti-proliferative effect of histamine. In some donors, histamine enhanced the percentage of IL-2 receptor positive cells. Stimulated PBMC from AIDS KS patients as a group, displayed a lower percentage of IL-2 receptor bearing cells, which was significantly increased by the addition of histamine even at concentrations as low as 10(-6) M and peaking at 10(-3) M. These findings indicate that histamine exerts its anti-proliferative effects on T cells by inhibiting IL-2 production, via blockade of IL-2 gene expression. In addition, histamine seems to exert immunomodulating effects on IL-2 receptor expression, particularly in those individuals with AIDS-KS.

Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.

Diphtheria toxin (DTx) is an extremely potent inhibitor of protein synthesis. Cell death has been generally accepted as a straightforward effect of translation inhibition. Using human U937 cells, we found that DTx intoxication leads to cytolysis; indeed, release of 51Cr- and 75Se-labeled proteins could be detected within 7 h. However, little or no cell lysis was observed over a 20-50-h period when human U937 cells were exposed to cycloheximide, amino acid-deficient medium, or metabolic poisons even though protein synthesis was rapidly inhibited to levels observed with DTx. Likewise, investigations with human K562 cells revealed full resistance to the cytolytic action of DTx over a 50-h period despite a severe reduction in translation activity. These observations establish that inhibition of protein synthesis per se is not sufficient to provoke cell lysis. A characterization of DTx-induced cytolysis revealed a long lag period (6-7 h) which could be shortened considerably by a short exposure to low pH. NH4Cl and metabolic poisons blocked the cytolytic action of DTx, indicating that endocytic uptake of toxin is required for lytic activity. Surprisingly, DTx also induced extensive internucleosomal degradation of cellular DNA, a characteristic feature of apoptosis or programmed cell death. DNA-fragmentation preceded cell lysis and did not occur in DTx-treated K562 cells or in U937 cells that were treated with the other protein synthesis inhibitors. From these observations, we conclude that DTx-mediated cytolysis is not a simple consequence of translation inhibition and that internucleosomal DNA fragmentation is a newly identified and relatively early step in the cytolytic pathway of DTx.

Amphotericin B-induced changes in renal membrane permeation: a model of nephrotoxicity.

As part of an investigation into the nephrotoxic effects of the polyene antibiotic Amphotericin B we have studied its effects on the ion permeability of purified renal brush border membrane vesicles. Membrane potentials were measured using a potential sensitive carbocyanine dye, and ion permeabilities were calculated from the constant field equation. Amphotericin B significantly altered the ionic permeability sequence of isolated membranes and caused a selectivity for increasing the permeation of anions. Permeability changes induced by 2.0 micrograms/ml Amphotericin B resulted in an estimated hyperpolarization of the membrane from -50 mV to -72 mV. In addition, the kinetic parameters of Na+ dependent transport of organic metabolites were examined. The maximum change in fluorescence was decreased significantly in the presence of Amphotericin B. These results suggest that the ionic state of the renal cell membrane is significantly altered by the presence of Amphotericin B.

Capacity of tumor necrosis factor to bind and penetrate membranes is pH-dependent.

Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4. A detailed examination of TNF-membrane interactions revealed that although TNF bound avidly to model membrane targets, no damage was generated under any condition tested. Binding of TNF, monitored with 125I-labeled as well as unlabeled protein, was enhanced at low pH. In the pH range tested (i.e., 4 to 8), target membrane permeability actually decreased in the presence of TNF. This membrane stabilization may be a consequence of TNF insertion into the target bilayer, a process we detected through use of an intramembranous photolabeling assay; interestingly, the efficiency of TNF insertion into membranes increased dramatically with decreasing pH. We conclude that native TNF does not cause pore formation directly and that its ability to induce cell lysis, as monitored by 51Cr release, is a consequence of some as yet obscure signaling event or intracellular activity. Parallel studies were carried out with diphtheria toxin, a protein with a more thoroughly characterized pH-dependent intoxification pathway. This toxin displayed acid-enhanced activities with both biologic and artificial targets.

Temperature dependence of membrane ion conductance analyzed by using the amphiphilic anion 5/6-carboxyfluorescein.

The rate of efflux of trapped 5/6-carboxyfluorescein from sealed lipid vesicles showed a marked dependence on (a) temperature, (b) phospholipid acyl chain composition, and (c) the nature of co-trapped counterions. When the dye was salted with sodium, at pH greater than 7, the rate of dye permeation showed a discrete maximum at the melting point of the lipid bilayer (Tc); in the case of membranes composed of dipalmitoylphosphatidylcholine, this discontinuity extended over a very broad temperature range, being detectable at least 10 degrees C above and below Tc. The peak in dye permeation rate was superimposed on a permeation profile that showed a simple exponential relationship to temperature. Studies with a homologous series of saturated lecithin bilayers revealed a consistent pattern of behavior: a logarithmic dependence of dye permeation rate on temperature with a superimposed discontinuity at Tc. For thin membranes (12-14-carbon acyl chains), the discontinuity was severe, exerting an influence over a very broad temperature range and leading to extremely high overall dye leakage rates. As the acyl chains were lengthened, the discontinuity became less pronounced, almost disappearing at a chain length of 20 carbons. In sharp contrast to these results, dye salted with N-methylglucamine [or with tris(hydroxymethyl)aminomethane] showed no efflux maximum at Tc, and base-line leakage rates were generally slower. When dye was salted with ammonium, efflux was too rapid to monitor, even at temperatures well below Tc. The results indicate that the rate of release of electrically charged dyes, such as 5/6-carboxyfluorescein, from sealed lipid vesicles can be tightly coupled to the counterion leakage rate and hence can provide an accurate and convenient assay of relative ion flux across phospholipid bilayers.

Conductance routes for protons across membrane barriers.

Simple phospholipid bilayers show a high level of permeability to protons; in spite of this fact, large proton gradients existing across such bilayers may decay very slowly. In sealed systems, the free movement of protons across a membrane barrier is severely restricted by the coincident development of a proton diffusion potential. Using the fluorescent weak acid N-[5-(dimethylamino)naphth-1-ylsulfonyl]glycine strongly buffered systems movement of the small number of protons giving rise to this electrical potential is insufficient to perturb the proton concentration gradient; significant flux of protons (and hence significant collapse of the concentration gradient) can only occur if protons traverse the membrane as part of an electroneutral complex or if there is a balancing flow of appropriate counterions. In both instances, proton flux is obligatorily coupled to the translocation of species other than protons. In weakly buffered systems, the small initial uncoupled electrogenic flux of protons may significantly alter the concentration gradient. This initial rapid gradient collapse caused by uncoupled electrogenic proton movements is then superimposed upon the residual collapse attributable to tightly coupled proton flux. The initial uncoupled electrogenic proton flux shows a temperature dependence very similar to that demonstrated for water permeation across simple lipid bilayers; upon cooling, there is a sharp decrease in flux at the temperature coinciding with the main gel-liquid-crystalline phase transition of the lipid. The coupled proton flux shows a markedly different temperature dependence with no dramatic change in rate at the phase transition temperature and strong similarity to the behavior previously seen with solutes known to be permeating as electrically neutral compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Close association between clathrin and the hydrophobic domain of boundary membranes in brain endocytic vesicles.

Purified bovine brain clathrin binds readily, in a pH-dependent fashion, to protein-free phospholipid bilayers. The association is tight and leads to inter-bilayer fusion, however, photolabeling studies using the amphiphilic photoreactive glycolipid 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine provide no evidence for direct insertion of clathrin into the central, hydrophobic domain of of these target membranes. In contrast, similar photolabeling studies of isolated, intact clathrin-coated vesicles show that, in these structures, clathrin is readily accessible to a probe which is known to reside preferentially within the hydrophobic domain of the membrane. The results are consistent with a natural requirement, by clathrin, for accessory proteins in order to effect membrane penetration.

Studies on the lethal hit stage of natural killer cell-mediated cytotoxicity. I. Both phorbol ester and ionophore are required for release of natural killer cytotoxic factors (NKCF), suggesting a role for protein kinase C activity.

Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC.

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients.

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.

The use of DNA-intercalating dye to monitor cell fusion and microinjection.

A conventional method for microinjection, using erythrocyte ghosts as the injection vector, has been modified to provide a protocol for the highly efficient delivery of small quantities of material into the cytoplasm of target cells. The technique is applicable for use with a variety of proteins, sugars, nucleotides and dyes. When the intercalating dye propidium iodide is included within the sealed ghosts their subsequent fusion with target cells can be continuously monitored by fluorescence spectroscopy, providing a convenient and sensitive parameter of cell-cell fusion. The protocol can be adapted for use with both adherent and non-adherent target cells, and can be used to monitor the relative effectiveness of a variety of fusogenic agents.

Permeability of lipid bilayers to water and ionic solutes.

The lipid bilayer moiety of biological membranes is considered to be the primary barrier to free diffusion of water and solutes. This conclusion arises from observations of lipid bilayer model membrane systems, which are generally less permeable than biological membranes. However, the nature of the permeability barrier remains unclear, particularly with respect to ionic solutes. For instance, anion permeability is significantly greater than cation permeability, and permeability to proton-hydroxide is orders of magnitude greater than to other monovalent inorganic ions. In this review, we first consider bilayer permeability to water and discuss proposed permeation mechanisms which involve transient defects arising from thermal fluctuations. We next consider whether such defects can account for ion permeation, including proton-hydroxide flux. We conclude that at least two varieties of transient defects are required to explain permeation of water and ionic solutes.

Phospholipid packing asymmetry in curved membranes detected by fluorescence spectroscopy.

There are distinct differences in the molecular packing of phospholipid molecules in the inner and outer membrane monolayers of small lipid vesicles; a small radius of curvature imparts an asymmetry to the interface between these two monolayers. I have used an amphiphilic fluorescent probe, N-[5-(dimethylamino)naphthalenyl-1-sulfonyl]glycine (dansylglycine), to determine if this asymmetry in molecular packing leads to the existence of different environments for fluorescent probes resident in the membrane. Dansylglycine is highly sensitive to the dielectric constant of its environment, and the fluorescence signal from membrane-bound dye is distinct from that in the aqueous medium. When dansylglycine is first mixed with vesicles, it rapidly partitions into the outer monolayer; the subsequent movement of dye into the inner monolayer is much slower. Because of the time lag between the initial partitioning and the subsequent translocation, it is possible to measure the emission spectrum from membrane-bound dye before and after translocation, thus distinguishing the two potential environments for dansylglycine molecules. In the outer membrane monolayer of small dipalmitoylphosphatidylcholine vesicles, dye fluorescence emission is maximal at 530 nm, corresponding to a dielectric constant of 7 for the medium surrounding the fluorophore. For dye in the inner monolayer, emission is maximal at 519 nm, corresponding to a dielectric constant of 4.7. The results suggest that water molecules are excluded more efficiently from the dye binding sites of the inner membrane monolayer than they are from those of the outer monolayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of human interleukin 2 produced from normal human peripheral blood mononuclear cells.

We have developed a simple protocol for the routine purification of essentially homogeneous human interleukin 2. The procedures applied include ammonium sulfate precipitation, ACA-54 gel filtration, ultrafiltration and chromatofocusing. The product has a molecular weight of 14 000, as determined by electrophoretic mobility, and is free of interleukin 1, interferon, granulocyte and monocyte stimulating factors, B cell growth factor and phytohemagglutinin. The method is efficient, rapid and reproduceable and provides a helpful method for preparation of IL-2 for biochemical and biological studies at moderate cost and without the use of complex equipment.