Response to and expression of amphiregulin by ovarian carcinoma and normal ovarian surface epithelial cells: nuclear localization of endogenous amphiregulin

Amphiregulin (AR) is a polypeptide growth regulator which has sequence homology to the epidermal growth factor-related family of ligands and contains putative nuclear targeting sequences. Human ovarian carcinoma cell lines and their normal counterparts, ovarian surface epithelial cells (OSEs), were assessed for their ability to respond to and express AR. Addition of exogenous AR (8-200 pM) inhibited the growth of 2 of 3 OSE specimens and 3 of the 6 carcinoma cell lines indicating that AR has the potential to inhibit the growth of normal cells, in addition to carcinoma cells. In contrast, concentrations of AR ranging from 1-5 nM stimulated the growth of all 3 of the OSEs and 4 of the 6 carcinoma cell lines. Immunocytochemical staining of the cells using antipeptide antibodies directed against residues 8-26 of AR indicated that all cells expressed AR and that the staining was localized to the nucleus. The nuclear staining of AR was concentrated in the nucleolus of the carcinoma cells, whereas the staining was diffuse in the nucleus of the OSEs. These results suggest that AR may play a growth regulatory role in the nucleus of cells and this role may be different in normal and malignant epithelial cells.     

Uptake and washout of borocaptate sodium and borono-phenylalanine in cultured melanoma cells: a multi-nuclear NMR study

The cellular uptake and washout of the two principal boron neutron capture therapy (BNCT) agents, borocaptate sodium (BSH) and borono-phenylalanine (BPA), were monitored on-line, noninvasively, using nuclear magnetic resonance (NMR) spectroscopy. The uptake and washout of inorganic borate (B(i)) was also followed for comparison. M2R mouse melanoma cells grown on polystyrene microspheres were perfused inside the NMR sample tube. (11)B NMR was used to detect the presence of B(i), BSH and BPA, and (19)F NMR was applied to detect fluorinated BPA ((19)F-BPA). The results revealed chemical modifications of BSH due to spontaneous formation of the borocaptate dimer, BSSB, in the culture medium. BPA readily formed a complex with glucose contained in the culture medium but was also converted in the cells to a yet unidentified compound in a reaction that probably involves the hydrolysis of BPA and the release of B(i). The cellular accumulation ratio for BPA was significantly higher than 1 and was also significantly higher than that for BSH. On the other hand, the cellular retention time observed for BSH was much longer than for BPA, indicating a strong trapping of BSH in cells.     

The effect of amphetamines on culture myotubes: Selective inhibition of protein synthesis

d-amphetamine sulfate, p-chloramphetamine and fenfluramine (10–100μM concentrations) were found to selectively inhibit protein synthesis in cultured chick myotubes. The most strongly inhibited cellular protein was a 34000 dalton polypeptide; myosin was affected to a smaller extent, while actin and tubulin were the least affected. 25μM of the drugs, or more, inhibited the incorporation of amino acid into proteins by 50%, while not reducing the acetylcholinesterase activity. The selective inhibition of protein synthesis may be one possible mechanism of the long-lasting effects of those drugs.     

Detection of light images by simple tissues as visualized by photosensitized magnetic resonance imaging

In this study, we show how light can be absorbed by the body of a living rat due to an injected pigment circulating in the blood stream. This process is then physiologically translated in the tissue into a chemical signature that can be perceived as an image by magnetic resonance imaging (MRI). We previously reported that illumination of an injected photosynthetic bacteriochlorophyll-derived pigment leads to a generation of reactive oxygen species, upon oxygen consumption in the blood stream. Consequently, paramagnetic deoxyhemoglobin accumulating in the illuminated area induces changes in image contrast, detectable by a Blood Oxygen Level Dependent (BOLD)-MRI protocol, termed photosensitized (ps)MRI. Here, we show that laser beam pulses synchronously trigger BOLD-contrast transients in the tissue, allowing representation of the luminous spatiotemporal profile, as a contrast map, on the MR monitor. Regions with enhanced BOLD-contrast (7-61 fold) were deduced as illuminated, and were found to overlap with the anatomical location of the incident light. Thus, we conclude that luminous information can be captured and translated by typical oxygen exchange processes in the blood of ordinary tissues, and made visible by psMRI (Fig. 1). This process represents a new channel for communicating environmental light into the body in certain analogy to light absorption by visual pigments in the retina where image perception takes place in the central nervous system. Potential applications of this finding may include: non-invasive intra-operative light guidance and follow-up of photodynamic interventions, determination of light diffusion in opaque tissues for optical imaging and possible assistance to the blind.     

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Specific Lipoplex-Mediated Antisense Against Bcl-2 in Breast Cancer Cells: A Comparison between Different Formulations

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3β[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms — either large unilamellar vesicles (～100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.