Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags

Reactive phosphorylcholine polymers, which can recognize biosynthetic cell-surface tags, were synthesized to control cell attachment. Human promyelocytic leukemia cells (HL-60) with unnatural carbohydrates as cell-surface tags were harvested by treatment with N-levulinoylmannosamine (ManLev). The attachment of ManLev-treated HL-60 cells to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with hydrazide groups was studied. HL-60 cells, which are nonadhesive, did not attach to any polymer surface without ManLev treatment. In contrast, ManLev-treated HL-60 cells attached to a poly[MPC-co-n-butyl methacrylate (BMA)-co-methacryloyl hydrazide (MH)] (PMBH) surface following 15 min of incubation. The cells that attached to the PMBH surface retained their native morphology and viability for 24 h of incubation. On the other hand, approximately half of the HL-60 cells that attached to the poly(BMA-co-MH) (PBH) surface died. These results suggest that MH units in the polymer act as anchors for cell attachment and MPC units help to preserve cell viability on a polymer surface. The coculture of ManLev-treated HL-60 and fluorescence-stained human uterine cervical cancer cells (HeLa) was carried out on polymer surfaces. ManLev-treated HL-60 cells specifically attached to the PMBH surface. In contrast, both HL-60 and HeLa cells were observed on the PBH surface. The control of cellular interactions with synthetic polymers may be useful for the future development of cell-integrated biosensors and biomedical devices.     

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.     

Conjugation of enzymes on polymer nanoparticles covered with phosphorylcholine groups

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.     

Nanoneedle Surface Modification with 2-Methacryloyloxyethyl Phosphorylcholine Polymer to Reduce Nonspecific Protein Adsorption in a Living Cell

Previously, we developed a new molecular delivery system to target single living cells by using atomic force microscope and ultrathin needle referred to as nanoneedle. This system delivers molecules into the cell by attaching them to the surface of nanoneedle. However, nonspecific protein adsorption on the nanoneedle surface inside the living cells limits the range of application of this system. In the present study, we focused on nonspecific protein adsorption onto the nanoneedle surface inside the cells and examined whether this protein adsorption was reduced by modifying the nanoneedle surface with a biocompatible phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. MPC polymer coating of the surface of silicon wafer reduced nonspecific adsorption of proteins from liver extracts and prevented the formation of clot-like protein aggregates. MPC polymer also decreased nonspecific adsorption of cytosolic protein onto the nanoneedle surface inside the living cell. On the other hand, MPC polymer showed no effect on nonspecific mechanical interaction between nanoneedle and the cell components. Surface modification with MPC polymer is a useful technique to modify the surface properties of nanoneedle.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Engineering novel cell surface receptors for virus-mediated gene transfer.

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.     

Phosphorylcholine Group-immobilized Surface Prepared on Polydimethylsiloxane Membrane by In Situ Reaction for Its Reduced Biofouling

To reduce interactions between biological molecules and the surface of microchip devices including the microchip, which should be conducted to improve sensitivity, reactivity, and the typical phospholipid polar group, the phosphorylcholine group-immobilized surfaces were prepared. The surface modification of polydimethylsiloxane (PDMS) was performed by in situ reaction during curing by cross-linking the PDMS prepolymers. Since it is known that 2-methacryloyloxyethyl phosphorylcholine (MPC) facilitates the preparation of biomedical polymers with excellent biocompatibility and antithrombogenicity, it was used as the reactant for surface modification. The MPC was coated on the glass substrate, and two-liquid-type PDMS prepolymers were then applied. During the curing process of the vinyl groups of poly(dimetylsiloxane-co-methylsiloxane) and poly(dimethylsiloxane-co-methylvinylsiloxane), the methacrylate group in MPC was attached onto the PDMS surface via a hydrosilyl group. Analysis of the surface characteristics by X-ray photoelectron spectroscopy and measurement of the surface contact angle revealed that the introduction of the phosphorylcholine group in the MPC unit on the surface induced hydrophilicity at the surface. Further, protein adsorption on the surface decreased with an increase in the number of phosphorylcholine groups. Based on these results, we concluded that the construction of the phosphorylcholine group-enriched surface on the PDMS substrate could be achieved by immobilization of MPC, and it may facilitate fabrication of biomedical devices, particularly microfluidic devices.     

A Round-bottom 96-well Polystyrene Plate Coated with 2-methacryloyloxyethyl Phosphorylcholine as an Effective Tool for Embryoid Body Formation

In this study, we proposed a culture method for forming embryoid bodies (EBs) from mouse embryonic stem (ES) cells using a round-bottom 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine (MPC plate). MPC is a phospholipid biocompatible polymer and prevents cells from adhering to the culture surface. The ES cells were seeded at 1000 cells per well in the MPC plate with 200 μl of medium. After 5 days of static incubation, a spherical cell aggregate termed EB was formed in a well. The size (diameter) of resulting EB was approximately 550 μm and it contained approx. 22,000 cells. It seems that the non-adhesiveness and the roundness of the well are important factors to form a good EB. Transferring the EBs to the attached differentiation culture, the EBs spread out and flattened, and the beating cells (cardiomyocytes) were effectively generated in the outgrowth of EBs. The round-bottom 96-well polystyrene plate coated with MPC is an effective tool for EB formation.     

Cell adhesion and morphology in porous scaffold based on enantiomeric poly(lactic acid) graft-type phospholipid polymers

Poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA) macromonomers were synthesized for preparation of a novel cytocompatible polymer. The cytocompatible polymer was composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and the enantiomeric PLLA (or PDLA) macromonomer. The degree of polymerization of the lactic acid in the PLLA and PDLA segments was designed to be ca. 20. The copolymer-coated surface was analyzed with static contact angle by water. From the result, the PLLA (or PDLA) segment and MPC unit were located on the coated surface, and the monomer unit in the copolymer was reconstructed by contacting water. Fibroblast cell culture was performed to evaluate cell adhesion on the coated surface, and the cell morphology was observed. The number of cell adhesion is correlated with the PL(D)LA content, and the cell morphology is correlated with the MPC unit content. The porous scaffold was prepared by the formation of a stereocomplex between the PLLA and PDLA, and the cell adhesion and following cell intrusion was then evaluated. The fibroblast cells adhered on the surface and intruded into the scaffold through the connecting pores after 24 h. The cell morphology became round shape from spreading with the decreasing PLLA (or PDLA) content in the copolymer. It is considered that the change in the cell morphology would be induced by the MPC unit as cytocompatible unit. These findings suggest that the porous scaffold makes it possible to have cytocompatibility and to produce three-dimensional tissue regeneration.     

Polymer conjugates of acridine-type anticancer drugs with pH-controlled activation

Acridines are potent DNA-intercalating anticancer agents with high in vivo anticancer effectiveness, but also severe side effects. We synthesized five 9-anilinoacridine-type drugs and their conjugates with biocompatible water-soluble hydrazide polymer carrier. All of the synthesized acridine drugs retained their in vitro antiproliferative properties. Their polymer conjugates were sufficiently stable at pH 7.4 (model of pH in blood plasma) while releasing free drugs at pH 5.0 (model of pH in endosomes). After internalization of the conjugates, the free drugs were released and are visible in cell nuclei by fluorescence microscopy. Their intercalation ability was proven using a competitive ethidium bromide displacement assay.     

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.     

Synthesis of novel folic acid-functionalized biocompatible block copolymers by atom transfer radical polymerization for gene delivery and encapsulation of hydrophobic drugs

Two synthetic routes to folic acid (FA)-functionalized diblock copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and either 2-(dimethylamino)ethyl methacrylate [DMA] or 2-(diisopropylamino)ethyl methacrylate [DPA] were explored. The most successful route involved atom transfer radical polymerization (ATRP) of MPC followed by the tertiary amine methacrylate using a 9-fluorenylmethyl chloroformate (Fmoc)-protected ATRP initiator. Deprotection of the Fmoc groups produced terminal primary amine groups, which were conjugated with FA to produce two series of novel FA-functionalized biocompatible block copolymers. Nonfunctionalized MPC-DMA diblock copolymers have been previously shown to be effective synthetic vectors for DNA condensation; thus, these FA-functionalized MPC-DMA diblock copolymers appear to be well suited to gene therapy applications based on cell targeting strategies. In contrast, the FA-MPC-DPA copolymers are currently being evaluated as pH-responsive micellar vehicles for the delivery of highly hydrophobic anticancer drugs.     

Anticancer potential of AgNPs synthesized using Acinetobacter sp. and Curcuma aromatica against HeLa cell lines: A comparative study

BackgroundBiogenic nanoparticles are gaining attention due to their low toxicity and numerous biomedical applications. Present study aimed to compare the potential anticancer activity of two biogenic silver nanoparticles (bAgNPs and pAgNPs) against human cervical cancer cell lines (HeLa).MethodsbAgNPs were synthesized using Acinetobacter sp. whereas pAgNPs were synthesized using aqueous root extract of Curcuma aromatica. Effect of these nanoparticles on HeLa cells viability was studied using MTT assay and colony formation assay. Anticancer potential was determined using fluorescence microscopy and flow cytometry studies. Bio-compatibility studies were performed against peripheral blood mononuclear cells (PBMCs).ResultsBoth the nanoparticles showed 50 % viability of peripheral blood mononuclear cells (PBMCs) when used at high concentration (200 μg/mL). IC₅₀ for bAgNPs and pAgNPs against HeLa cells were 17.4 and 14 μg/mL respectively. Colony formation ability of Hela cells was reduced on treatment with both nanoparticles. Acridine orange and ethidium bromide staining demonstrated that bAgNPs were cytostatic whereas pAgNPs were apoptotic. JC-1 dye staining revealed that the mitochondrial membrane potential was affected on treatment with pAgNPs while it remained unchanged on bAgNPs treatment. Flow cytometry confirmed cell cycle arrest in HeLa cells on treatment with nanoparticles further leading to apoptosis in case of pAgNPs. About 77 and 58 % HeLa cells were found in subG1 phase on treatment with bAgNPs and pAgNPs respectively. bAgNPs showed cytostatic effect on HeLa cells arresting the cell growth in subG1 phase, whereas, pAgNPs triggered death of HeLa cells through mitochondrial membrane potential impairment and apoptosis.ConclusionOverall, bAgNPs and pAgNPs could be safe and showed potential to be used as anticancer nano-antibiotics against human cervical cancer cells.     

AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells

The apoptosis of cancer cells is associated with changes in the important cell properties including morphology, surface roughness and stiffness. Therefore, the changes in morphology and biophysical properties can be a good way of evaluating the anticancer activity of a drug. This study examined the effect of paclitaxel on the properties of Ishikawa and HeLa cells using atomic force microscopy (AFM), and the relationship between the changes in morphology and the biophysical properties and apoptosis was discussed. The viability and proliferation of the cells were analyzed using the methylthiazol tetrazolium (MTT) method and a TUNEL assay to confirm cellular apoptosis due to a paclitaxel treatment. AFM observations clearly showed the apoptotic morphological and biophysical changes in Ishikawa and HeLa cells. After the paclitaxel treatment, the cell membrane was torn and holed, the surface roughness was increased, and the stiffness was decreased. These changes were observed more apparently after a 24 h treatment and in Ishikawa cells compared to HeLa cells. The MTT and TUNEL assays results revealed the Ishikawa cells to be more sensitive to paclitaxel than HeLa cells and definite apoptosis occurred after a 24 h treatment. These results showed good agreement with the AFM results. Therefore, research on the morphological and biophysical changes by AFM in cancer cells will help to evaluate the anticancer activities of the drugs.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting.

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.     

Bioconjugated phospholipid polymer biointerface for enzyme-linked immunosorbent assay

This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer--poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surface maintained residual activity after long-term storage. This surface affords a low background without requiring blocking treatment and is suitable for immobilized antibodies.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.     

Selective biorecognition and preservation of cell function on carbohydrate-immobilized phosphorylcholine polymers

To obtain synthetic materials capable of selectively recognizing proteins and cells, and preserving their functions, biomembrane mimetic polymers having a phospholipid polar group and carbohydrate side chains were designed. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-2-lactobionamidoethyl methacrylate (LAMA)] (PMBL) was synthesized and coated on substrates by solvent evaporation. Selective binding of galactose-recognized lectin, RCA120, to a PMBL surface was investigated by measurement of surface plasmon resonance. The binding of RCA120 to the PMBL surface was confirmed by a remarkable change in resonance angle. The apparent affinity constant of RCA120 to PMBL3.0 (3.0 mol % LAMA unit in the feed) per LAMA unit was 2.77 x 10(5) M(-1). When a glucose-recognized lectin, concanavalin A, was in contact with PMBL, no change in the resonance angle was observed, and any nonspecific fouling of protein on PMBL was effectively reduced. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on polymer surfaces. On poly(BMA) (PBMA), many adherent cells were observed and were well-spread with monolayer adhesion, but cell adhesion was reduced on poly(MPC-co-BMA) (PMB). HepG2 adhesion was observed on PMBL because the cell has ASGPRs; the number of cells adhering to the PMBL polymer surfaces increased with an increase in the density of galactose residues on the surface. In contrast, adhesion of NIH-3T3 cells to PMBL was reduced in a manner similar to that on PMB because the NIH-3T3 cells did not have ASGPRs. Cell adhesion to the PMBL surface was well-regulated by ligand-receptor interactions. Furthermore, some of the cells adhering to the PMBL surface had a spheroid form, and similarly shaped spheroids were scattered on the surface. Although poly(BMA-co-LAMA) (PBL) has galactose residues, the adherent cells were spread in a manner similar to those on PBMA. The MPC units in PMBL contribute to make a spheroid formation of HepG2 cells. The amount of albumin secreted from a cell was compared with the chemical structure of the substrate. The spheroid shaped cells cultured on the PMBL surface secreted much more albumin than did the spreading cells that adhered to the PBMA. In conclusion, the biomembrane mimetic carbohydrate-immobilized phosphorylcholine polymers produced a suitable surface for biorecognition and preservation of cell function.     

Control of nanobiointerfaces generated from well-defined biomimetic polymer brushes for protein and cell manipulations

To better understand protein/material and cell/material interactions at the submolecular level, well-defined polymer brushes consisting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) on silicon wafers were prepared by atom transfer radical polymerization (ATRP). Silicon wafers were treated with 3-(2-bromoisobutyryl)propyl dimethylchlorosilane (BDCS) to form a monolayer that acts as initiators for ATRP. Silicon-supported BDCS monolayers were soaked in a methanol/water mixture solution containing Cu(I)Br, bipyridine, and a sacrificial initiator. After MPC was added to the solution, ATRP was carried out for 18 h. The molecular weight and thickness of the PMPC brush layer on the silicon surface increased with an increase in the polymerization time. The dense polymer brushes were obtained by the "grafting from" system. By selective decomposition of the BDCS monolayer by UV light-irradiation, the PMPC brush region and the sizes were well controlled, resulting in fabricating micropatterns of the PMPC brushes. When the thickness of the PMPC brush layer was greater than 5.5 +/- 1.0 nm (3 h polymerization), serum protein adsorption and fibroblast adhesion were effectively reduced, i.e., proteins and cells could recognize such thin polymer brushes on the surface. In addition, the density of the adherent cells on the patterned PMPC brush surface could be controlled by changing the size of the pattern.     

Differential targets and subcellular localization of antitumor alkyl-lysophospholipid in leukemic versus solid tumor cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.