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Two species from the families Acipenseridae and Polyodontidae, Russian sturgeon (Acipenser gueldenstaedtii, Brandt and Ratzeberg, 1833, functional tetraploid) and American paddlefish (Polyodon spathula, Walbaum 1792, functional diploid) were hybridized. The hybridization was repeated using eggs from three sturgeon and sperm from four paddlefish individuals. Survival in all hybrid family groups ranged from 62% to 74% 30 days after hatching. This was the first successful hybridization between these two species and between members of the family Acipenseridae and Polyodontidae. Flow cytometry and chromosome analysis revealed two ploidy levels in hybrids. The chromosome numbers of the hybrids ranged between 156&ndash, 184 and 300&ndash, 310, in &ldquo, functional&rdquo, triploids and &ldquo, pentaploids, respectively. The hybrid origin and the ploidy levels were also confirmed by microsatellite analyses. In hybrids, the size and the number of dorsal and ventral scutes correlated with the ploidy levels as well as with the calculated ratio of the maternal and paternal chromosome sets. An extra haploid cell lineage was found in three hybrid individuals irrespective of the ploidy level, suggesting polyspermy. Although the growth performance showed high variance in hybrids (mean: 1.2 kg, SD: 0.55), many individuals reached a size of approximately 1 kg by the age of one year under intensive rearing conditions.

We studied the effect of different magnitudes (7000 PSI (48.26 MPa), 8000 PSI (55.16 MPa), and 9000 PSI (62.05 MPa)) of hydrostatic pressure on the ploidy of pikeperch larvae. Pressure shock was applied 5 min after the fertilization of eggs at a water temperature of 14.8 ± 1 °C. A 7000 PSI pressure shock was applied for 10 or 20 min, while 8000 and 9000 PSI treatments lasted for 10 min. Each treatment with its respective control was completed in triplicate, where different females’ eggs served as a replicate. In the treatment groups exposed to 7000 PSI for 10 min, only diploid and triploid larvae were identified, while 2n/3n mosaic individuals were found after a 20-min exposure to a 7000 PSI pressure shock. The application of 8000 or 9000 PSI pressure shocks resulted in only triploid and mosaic individuals. Among larvae from eggs treated with 8000 PSI, three mosaic individuals with 2n/3n karyotype were identified (4.0 ± 6.9%), while a single (2.0 ± 3.5%) 1n/3n mosaic individual was found in the 9000 PSI-treated group. To our knowledge, this is the first report that demonstrates the induction of a haplo-triploid karyotype by hydrostatic pressure shock in teleost fish. The dominance of triploid individuals with a reasonable survival rate (36.8 ± 26.1%) after 8000 PSI shock supports the suitability of the hydrostatic pressure treatment of freshly fertilized eggs for triploid induction in pikeperch.

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[18F]fluoro-2-deoxy-D-glucose (18FDG),11C-methionine, 3′-deoxy-3′-[18F]fluorothymidine (18F-FLT), and [18F]fluoroazomycin-arabinofuranoside (18FAZA) forin vivofollow-up of the efficacy of chemotherapy in both Pgp positive (Pgp+) and negative (Pgp−) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp+and Pgp−A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp+tumors. Our results demonstrate that18FDG,18F-FLT,18FAZA, and11C-methionine are suitable PET tracers for the diagnosis andin vivofollow-up of the efficacy of tumor chemotherapy in both Pgp+and Pgp−human tumor xenografts by miniPET.

Retinoids – derivatives of vitamin A – are important cell permeant signaling molecules that regulate gene expression through activation of nuclear receptors. P-glycoprotein (Pgp) and ABCG2 are plasma membrane efflux transporters affecting the tissue distribution of numerous structurally unrelated lipophilic compounds. In the present work we aimed to study the interaction of the above ABC transporters with retinoid derivatives. We have found that 13-cis-retinoic acid, retinol and retinyl-acetate inhibited the Pgp and ABCG2 mediated substrate transport as well as the substrate stimulated ATPase activity of these transporters. Interestingly, 9-cis-retinoic acid and ATRA (all-trans retinoic acid), both are stereoisomers of 13-cis-retinoic acid, did not have any effect on the transporters’ activity. Our fluorescence anisotropy measurements revealed that 13-cis-retinoic acid, retinol and retinyl-acetate selectively increase the viscosity and packing density of the membrane. Thus, the mixed-type inhibition of both transporters by retinol and ABCG2 by 13-cis-retinoic acid may be the collective result of direct interactions of these retinoids with the substrate binding site(s) and of indirect interactions mediated by their membrane rigidifying effects.

P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided.

P-glycoprotein (Pgp, ABCB1) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing the phenomenon of multidrug resistance. It has been shown earlier that the combined application of a class of Pgp modulators (e.g. cyclosporine A and SDZ PSC 833) used at low concentrations and UIC2 antibody is a novel, specific, and effective way of blocking Pgp function (Goda et al., 2007). In the present work we study the UIC2 antibody mediated Pgp inhibition in more detail measuring the accumulation of tumor diagnostic radiotracers, 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) and [(99m)Tc]hexakis-2-methoxybutyl isonitrile ((99m)Tc-MIBI), into Pgp(+) (A2780AD) and Pgp(-) (A2780) human ovarian carcinoma cells. Co-incubation of cells with UIC2 and cyclosporine A (CSA, 2μM) increased the binding of UIC2 more than 3-fold and reverted the rhodamine 123 (R123), daunorubicin (DNR) and (99m)Tc-MIBI accumulation of the Pgp(+) 2780AD cells to approx. the same level as observed in Pgp(-) cells. Similarly, 50μM paclitaxel (Pacl) increased UIC2 binding, and consequently reinstated the uptake of R123, DNR and (99m)Tc-MIBI into the Pgp(+) cells. Blocking Pgp by combined treatments with CSA+UIC2 or Pacl+UIC2 also decreased the glucose metabolic rate of the A2780AD Pgp(+) cells measured in (18)FDG accumulation experiments suggesting that the maintenance of Pgp activity requires a considerable amount of energy. Similar treatments of the A2780 Pgp(-) cells did not result in significant change in the R123, DNR, (99m)Tc-MIBI and (18)FDG accumulation demonstrating that the above effects are Pgp-specific. Thus, combined treatment with the UIC2 antibody and Pgp modulators can completely block the function of Pgp in human ovarian carcinoma cells and this effect can be followed in vitro by using tumor-diagnostic radiotracers, (99m)Tc-MIBI and (18)FDG.

We studied how very short (10–40 min) incubation with anthracycline derivatives modifies the accumulation of PET tumor-diagnostic radiotracers in cancer cells. The human ovarian A2780 and A2780AD, human B lymphoid JY, human epidermoid KB-3-1 and KB-V-1, and smooth muscle DDT1 MF-2 cells were pre-incubated with daunorubicin and doxorubicin, and the uptake of [18F]FDG and [11C]choline was measured. Anthracycline treatment decreased remarkably the [11C]choline accumulation in a concentration dependent manner, while it did not modify significantly the [18F]FDG uptake of the cells.

P-glycoprotein (Pgp), coded for by the mdr1 gene, is one of the ABC transporters held responsible for the phenomenon of multidrug resistance (mdr), which is reflected by a rapidly escalating failure of chemotherapy with different classes of cytotoxic agents: anthracyclins, vinca alkaloids, taxanes, epipodophylotoxins. Although overcoming resistance conveyed by Pgp alone may not be sufficient for reaching effective treatment, the abundance of observations available for this paradigmatic multidrug transporter at both in vitro and in vivo setting is a tempting ground for an updated assessment of the main currents of mdr research. In this review we attempt to help keep track of the features of Pgp-mediated drug transport that serve as the major starting points for ongoing efforts of mdr reversal. We will analyze the slowly narrowing gaps that prevail between our ever increasing understanding at the protein, cell and organism level, focusing on the molecular interactions involving Pgp.

The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.

AIM To study how paclitaxel treatment modifies the accumulation of tumor-diagnostic radiotracers in P-glycoprotein (P-gp) positive and negative cancer cells. METHODS The accumulations of different P-gp substrates, including rhodamine 123, daunorubicin and [(99m)Tc]hexakis-2-methoxybutyl isonitrile ((99m)Tc-MIBI), were measured in P-gp-positive (A2780AD) and P-gp-negative human ovarian carcinoma cells (A2780) and JY human lymphoid B cells. The uptakes of the tumor-diagnostic tracers (11)C-choline and 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) were measured in the same cell lines. The P-gp expression and function were demonstrated by flow-cytometry. RESULTS The (18)FDG measurements revealed that the glucose metabolic rate was significantly higher (p

A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing>or=1 microM Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3',4'-dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 microM, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50=2.44 microM). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role.

Background A novel flow cytometric assay has been described in an accompanying report (Gombos et al., Methods The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non–raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. Results The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and GM1-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. Conclusions By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels. © 2004 Wiley-Liss, Inc.

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3 0 -t o -5 0 - exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5 0 protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to

P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes con- formational changes in the presence of substrates/modula- tors, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclo- sporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished (Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & SzaboJr, G. (2001) Eur. J. Biochem. 268, 2416-2420). To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleo- tides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp- expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in re- sponse to direct modulation of the ATP-binding site, con- firming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.

cystic fibrosis transmembrane conductance regulator(CFTR, ABCC7) is a member of the human ABC protein fam-ily. The characteristic feature of ABC proteins is the similararchitecture of the ATP binding sites and the common mecha-nism of ATP binding and hydrolysis. In contrast to most ofthe ABC proteins, CFTR is not an active transporter, rather itis a phosphorylation and ATP hydrolysis gated chloride ionchannel and channel regulator (1,2). Mutations in both copiesof the CFTR gene may lead to a heritable genetic disease, i.e.cystic fibrosis (CF) with variable severity depending on thesite of the mutation. Certain mutations alter biogenesis or cel-lular processing of the CFTR protein, while others affect itschannel activity. Chemical chaperons, also called correctors(e.g., VX-809) can partially rescue the misprocessing, mostlikely by improving the folding of the protein at the endoplas-mic reticulum (2). Activators of CFTR may function throughelevating cytosolic cAMP (promoting CFTR phosphoryla-tion), inhibiting phosphatase activity (thus blocking CFTR de-phosphorylation), and/or interacting directly with the channelprotein (1). Since CFTR is expressed in the epithelial cells ofseveral organs, CF is a multi-organ disease that affects thesinopulmonary and male urogenital systems, alters pancreaticand biliary secretion, therefore the current average lifespan ofCF patients is approximately 40 years of age (1,2). In mostcases, the primary cause of early onset of death is the progres-sion of lung disease (2,3). The mechanisms by which CFTRmutations cause lung disease in CF patients are not fullyunderstood. It may include altered ion and water transportacross the airway epithelium and aberrant inflammatory andimmune responses to pathogens within the airways (3,4).Several techniques have been developed to study CFTRfunction or to search for CFTR activators or modulatorsincluding halide sensitive fluorescent dyes, electrophysiologi-cal approaches such as patch clamp, short-circuit measure-ments in Ussing chambers and influx or efflux measurementsapplying radioactive ions. In addition to the above techniquesa halide sensitive mutant form of yellow fluorescent protein(YFP) has been also utilized to probe CFTR function meas-uring the iodide-mediated quenching rate of YFP, since theopen state of CFTR is also permeable to iodide ions (5). YFPfluorescence intensity is both dependent on the intracellulariodide concentration and YFP expression level. Therefore, flu-orescence intensities measured in the presence of iodideshould be normalized to YFP fluorescence intensities meas-ured in the absence of iodide to decrease experimental vari-ability related to the differences in sensor expression levelamong individual cells.However, normalization to the un-quenched YFP fluores-cence intensity is problematic especially in flow cytometricassays. The co-expression of a halide insensitive fluorescentprotein can overcome the above problem as was demonstratedby Vijftigschild et al. published in the current issue of Cytome-try Part A (page 576). When the two fluorescent proteins wereexpressed as a fusion protein formation of aggregates wasobserved. To avoid this problem the two proteins were con-nected by an auto-cleavable polypeptide chain (6) resulting inphysically not connected proteins. Theoretically, the expres-sion level of the two proteins may change upon time due to

P-glycoprotein (Pgp) is a transmembrane protein that actively exports lipophilic chemotherapeutics from the cells causing multidrug resistance. Pgp molecules are partially localized in TX-100-resistant rafts, and the activity of the transporter is highly sensitive to the presence of cholesterol. To better understand these relationships, the influence of membrane cholesterol content on Pgp function, as measured via calcein accumulation, was studied in correlation with changes elicited in membrane structure. Membrane cholesterol was modulated by heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) and the cholesterol inclusion complex of DIMEB (Chol-DIMEB). Changes in membrane cholesterol level were reflected by alterations in the overall lipid packing as measured by Merocyanine 540 (MC540) staining and were also accompanied by changes in the raft association of the pump. DIMEB and Chol-DIMEB treatments have also lead to increased permeability of the cell membrane in both directions, raising the possibility that the effects on pumping efficiency reflect leakage of ATP also from the non-permeabilized cells. However, the treatments did not influence the intracellular ATP levels of the non-permeabilized cells. Our data suggest that Pgp inhibition by cyclodextrin treatments arises through modulation of its membrane microenvironment, rather than as a result of concomitant cytotoxicity.

Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S–S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S–S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S–S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S–S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at ≈50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia ( MLL ) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining ≈50-kbp domains.

P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 approximately 20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp+ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.

Background: Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA–protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi-quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics. Methods: We have established a flow-cytometry-based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads. Results: In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down-regulation of TGM2 mRNA expression. Conclusions: This method that we named ChIP-on-beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow-cytometric laboratory. © 2006 International Society for Analytical Cytology

MDR1-associated P-glycoprotein-dependent multidrug resistance is a common cause of chemotherapy failure in patients with advanced ovarian cancer. Here, we describe a clinical method for simultaneously assessing the expression and function of the MDR1/Pgp in tumour cells from ascites of patients with malignant ovarian carcinoma.Cells from ascites from 35 patients were collected. The expression and function of Pgp were detected by flow cytometry. For functional study, rhodamine 123 was used.Using the Pgp-specific UIC2 and MM6.15 antibodies, we demonstrated the presence of Pgp in 10-79% (38.9+/-20, 7; n=35) of the CA 125-positive cell subpopulations. The results of the functional assay showed strong correlation with the level of Pgp expression (r=0.976; p=3.2x10(-5)).Direct detection of the expression level and function of MDR1/Pgp in the ascites provide useful information for the more efficient treatment of malignant diseases by proper adjustment of the chemotherapeutic protocol.

Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method.Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents.Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach.A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.

A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents.The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping.By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.

P-glycoprotein (Pgp) is one of the ABC transporters responsible for the multidrug resistance of cancer cells. The conformational changes of Pgp that occur in the presence of substrates/modulators or ATP depletion are accompanied by the up-shift of UIC2 monoclonal antibody (mAb) binding. In the case of cyclosporin A, vinblastine or valinomycin, this up-shift was found to be concomitant with the near-complete suppression of labeling with other mAbs specific for Pgp epitopes overlapping with UIC2, while pre-treatment with verapamil or Tween 80 brings about a modest suppression. Here we have extended these observations to 44 Pgp interacting agents, and found that only 8 fall into the cyclosporin-like category, inducing a conformational state characterized by the complete UIC2 dominance. The rest of the drugs either did not affect antibody competition or had a modest effect. Thus, Pgp substrates/modulators can be classified into distinct modalities based on the conformational change they elicit.

P-glycoprotein (Pgp) is an ABC (ATP binding cassette) transporter that is often overexpressed in tumours, contributing significantly to their multidrug resistance. In this study, we explored whether the radiotracers used in tumour diagnostics can be used for in vivo visualisation of Pgp-related multidrug resistance. We also examined the effects of different Pgp modulators on the accumulation of these radioligands in tumours with or without Pgp expression. In a SCID BC-17 mouse model, cells of the drug-sensitive KB-3-1 (MDR(-)) and the KB-V1 Pgp-expressing (MDR(+)) human epidermoid carcinoma cell lines were inoculated to yield tumours in opposite flanks. For in vivo scintigraphic (biodistribution) and positron emission tomography (PET) examinations, the mice were injected with technetium-99m hexakis-2-methoxybutylisonitrile ((99m)Tc-MIBI), carbon-11 labelled methionine and fluorine-18 fluoro-2-deoxy- d-glucose ((18)FDG). For validation, in vitro cell studies with (99m)Tc-MIBI,( 99m)Tc-tetrofosmin, [(11)C]methionine and (18)FDG were carried out using a gamma counter. The expression and function of the MDR product were proved by immunohistochemistry and spectrofluorimetry. (99m)Tc-MIBI uptake was significantly lower in KB-V1 cells as compared with KB-3-1-derived tumours in vivo (Pgp(+)/Pgp(-) =0.61+/-0.13; P0.01) and cells in vitro (Pgp(+)/Pgp(-) =0.08+/-0.01; P0.001).()Cyclosporin A reversed (99m)Tc-MIBI uptake in the Pgp+ cells, while verapamil failed to modify it. (18)FDG uptake was significantly higher in KB-V1 tumours (Pgp(+)/Pgp(-) =1.36+/-0.05; P0.01) and cells (Pgp(+)/Pgp(- )=1.52+/-0.12; P0.001). Whereas cyclosporin A eliminated the difference between FDG uptake in MDR(+) and MDR(-) cell lines, verapamil significantly increased it. When the animals were treated with verapamil, the ratio of (99m)Tc-MIBI uptake in the MDR(+) tumours to that in the MDR(-) tumours decreased to 0.38+/-0.05 ( P0.01), while the ratio of (18)FDG uptake increased to 2.1+/-0.3 ( P0.001). There were no significant differences in the [(11)C]methionine uptake in the MDR(+) and MDR(-) tumours and cell lines, nor was [(11)C]methionine accumulation modified by cyclosporin A. Parallel administration of (18)FDG and (99m)Tc-MIBI combined with verapamil treatment seems to be a good candidate as a non-invasive marker for the diagnosis of MDR-related Pgp expression in tumours.

P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E.Szabó Jr, G. (2001) Eur. J. Biochem. 268, 2416-2420]. To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp-expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP-binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.

SummaryWe reported earlier that neonatal monocyte-derived macrophages (MDM) could not be fully activated with IFN-γ, a finding that could not be attributed to lower expression of IFN-γ receptors on the neonatal cells. In this study we explored elements of IFN-γR-mediated signalling in cord monocytes and MDM. Intracellular expression of STAT-1 was analysed by flow cytometry. We have assessed phosphorylation of STAT-1 by using MoAbs that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis. Using MoAbs against the native form of STAT-1 revealed comparable expression of this protein in cord and adult cells (both monocytes and MDM). However, STAT-1 phosphorylation in response to IFN-γ was significantly decreased in neonatal monocytes (P 5 for each). These data suggest deficient cytokine-receptor signalling in neonatal mononuclear phagocytes exposed to IFN-γ. We propose that decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.

Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal ("re-reversal"), exhibits a sharp extracellular potassium concentration ([K+(0)]) dependence. It is observed at [K+(0)]2-4 mM and not at [K+(0)] greater than or equal to 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that "re-reversal" is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+(0)]-dependence similar to that of the "re-reversal." These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells.

The intracellular pH (pH(i)) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-V1/KB-3-1 system in this paper. The possible role of an elevated pH(i) in Pgp-related MDR has been investigated by shifting back the pH(i) of the MDR+ cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked delta pH(i) on relative daunorubicin (DNR) accumulation was similar in the case of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pH(i) on Pgp pumping efficiency. However, an indirect connection between pH(i) regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR+ cells.