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S Orlowski

Publications and source records attributed to S Orlowski.

At least 37 records · Page 2Linked to original sources
PubMed1994

Involvement of membrane bleomycin-binding sites in bleomycin cytotoxicity.

The authors have recently shown the existence of bleomycin (BLM)-binding sites at the surface of DC-3F cells. In order to study the involvement of these sites in the sensitivity of the cells to bleomycin several BLM-resistant cell lines from DC-3F cells were analysed. These mutants were obtained by electrotransfection of the Sh ble gene (D/BlmI cells) or the Sh ble-beta Gal fusion gene (D/BlmII cells) and/or by continuous culture in the presence of BLM (D/BlmIR and D/Blm40 cells). The resistance levels of the D/BlmII and D/Blm40 cells were 50- and 22-fold, respectively, determined at the EC50 level. The D/BlmI cells were only 2-fold resistant, whereas D/BlmIR cells were so resistant that almost no cytotoxicity was detected up to 200 microM BLM external concentration. Electropermeabilization was used in an attempt to bypass the plasma membrane of the cells and permit the distinction between internal resistance and membrane resistance. The former was observed when the products of the transfected genes were present. With respect to membrane resistance, differences were detected in the number of BLM-binding sites in several mutant cell lines, which could account for the differences in cell sensitivity to BLM. This suggests that the BLM-binding sites found at the cell surface may play a crucial role in BLM internalization and consequently in its cytotoxicity.

Animals↗
PubMed1994

Electropermeabilization of cells in tissues assessed by the qualitative and quantitative electroloading of bleomycin.

Using cells in suspension, electropermeabilization is a technique extensively used to transfect living cells or to introduce a variety of compounds inside the cells. Here we demonstrate the reality of the tissue electropermeabilization using qualitative and quantitative determinations of the electroloading of bleomycin considered as an nonpermeant molecule that serves as an indicator of the permeabilization. In tissues, cell electropermeabilization is achieved for electric field intensities lower than those necessary to permeabilize the same cells in suspension. We also emphasize the importance of the geometry of the electric field lines defined by the electrodes for permeabilizing a whole tissue, for example a tumor.

Animals↗
PubMed1993

Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial.

BACKGROUND: Electrochemotherapy is a new antitumor treatment consisting of electrical pulses administered to the tumor several minutes after intravenous injection of bleomycin. In mice, important antitumor effects were observed on subcutaneously transplanted tumors and on spontaneously occurring mammary carcinomas. Cures were obtained after one single treatment combining bleomycin and electric pulses. In humans, permeation nodules seemed an adequate oncologic situation to assay this new procedure. The authors report the first Phase I-II trial of electrochemotherapy. METHODS: Eight patients with 40 permeation nodules of head and neck squamous cell carcinomas were treated with 10 mg/m2 bleomycin intravenous bolus, followed by four or eight short (100 microseconds) and intense (1300 V/cm) pulses administered through two external electrodes located on each side of the treated nodule. RESULTS: An instantaneous painless contraction of the underlying muscles was regularly observed. Neither local nor general side effects were observed, and electrochemotherapy was well tolerated. In addition, a clear local antitumor efficacy was found: 23 (57%) nodules were in clinical complete response within a few days. CONCLUSION: The absence of toxicity, the good tolerance by the patients, and the net antitumor effects observed are encouraging for additional electrochemotherapy developments in clinical oncology.

Adult↗
PubMed1993

Absence of cooperativity for MgATP and verapamil effects on the ATPase activity of P-glycoprotein containing membrane vesicles.

Purified membrane vesicles were prepared from Chinese Hamster lung fibroblasts expressing high amounts of P-glycoprotein (P-gp), which is responsible for the multidrug resistance. P-gp ATPase activity, characterized in the presence or absence of verapamil, had a Michaelian behavior for its MgATP dependence. Thus only one MgATP molecule should be sufficient for the catalytic cycle. With increasing verapamil concentrations, a bell-shape curve was observed for ATPase activity, with half-activation and -inhibition concentrations of 1.2 microM and 490 microM, respectively. No cooperativity for verapamil was detected. These results strongly suggest that P-gp functions as an active transporter, with a coupling stoichiometry of one MgATP molecule hydrolysed for one verapamil molecule transported.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Strontium binding to sarcoplasmic reticulum Ca(2+)-ATPase. Spectroscopic differentiation of the substeps involved.

We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca(2+)-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr(2+)-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr2+ concentrations than the Sr(2+)-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr(2+)-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr(2+)-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.

Binding Sites↗
PubMed1993

Cell electropermeabilization: a new tool for biochemical and pharmacological studies.

Cell electropermeabilization is the transient permeabilization of the plasma membrane by means of short and intense electric pulses. Under optimized conditions, electropermeabilization is compatible with cell survival. It provides a direct access into the cytosol to ions, small molecules, exogenous drugs and macromolecules. As cells remain functional, a large variety of cell biology questions can be addressed. Such 'in situ biochemistry' opens new possibilities beside the more classical studies dealing with unpermeabilized cells or subcellular extracts. Electropermeabilization also allows pharmacological studies with cells, cultured monolayers and in vivo tissues as well as the design of drug controlled-release systems.

Animals↗
PubMed1992

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.

Animals↗
PubMed1992

[Potentiation of the antitumoral effect of electrochemotherapy by immunotherapy with allogeneic cells producing interleukin 2].

Electrochemotherapy (ECT) is a new antitumour treatment which consists of delivering electric pulses to the tumour a few minutes after an intravenous injection of bleomycin. The antitumour efficacy of ECT is increased by local injections of interleukin 2 (IL2) in the oedema which appears at the site of the treated tumours. We have shown that tumour cells inoculated in syngeneic mice are rejected if IL2 secreting allo- or xenogeneic cells are co-injected with tumour cells. We report here the large increase of ECT therapeutical efficacy when allogeneic cells secreting IL2 are injected into the peri-tumoural oedema.

Animals↗
PubMed1991

Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture.

We observed previously in vitro that the cytotoxicity of bleomycin (BLM), an anticancer drug in current use, was greatly potentiated by exposing cultured cells to appropriately chosen electric pulses. We then showed in vivo, on tumor-bearing mice, that the same electric pulses also potentiated the antitumoral activity of BLM. In the present work, we demonstrate on DC-3F cells in vitro, that this potentiation is closely related to cell electropermeabilization and the consequent direct internalization of BLM molecules in the cytosol. The survival response curve (SRC) of the electropermeabilized (EP) cells exposed to BLM (plotted as logarithm of survival versus external drug concentration) shows a linear pattern usual for the SRCs of intact cells exposed to current cytotoxic drugs, though in the nanomolar range of concentrations. We have succeeded in determining the relation between BLM cytotoxicity on EP cells and the number of electroloaded BLM molecules per cell (that is the average number, per cell, of BLM molecules internalized into the cytosol). We conclude that (1) BLM molecules possess very intense cytotoxic activity which in non-EP cells is drastically limited by the intact plasma membrane; and (2) in these intact cells, the plasma membrane is responsible for the unusual upward concave curvature of the SRC resulting from exposure to BLM.

Animals↗
PubMed1991

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.

Animals↗
PubMed1991

The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen.

Using rapid filtration, we investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca2+ ions transported by the Ca(2+)-dependent ATPase of these vesicles. Release rates at 20 degrees C were measured by three methods, with vesicles previously made leaky with an ionophore. First, we measured the rate at which 45Ca2+ bound to ATPase approached its steady-state level after addition of ATP to the 45Ca(2+)-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of 45Ca2+ dissociation from phosphorylated ATPase. Second, we measured the kinetics of 45Ca2+ dissociation from phosphorylated ATPase in a "chase" experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. We found that these kinetics were essentially monophasic. Moreover, when they were measured in the presence of a high concentration of calcium, designed to saturate the low-affinity calcium transport sites on the lumenal side of the ATPase, they only departed slightly from monophasic behavior, irrespective of the experimental pH (pH 6, 7, or 9). This small perturbation by high calcium concentrations of the observed dissociation kinetics was attributed to ADP-facilitated rapid exchange of 40Ca2+ for Mg2+ at the catalytic site of phosphorylated ATPase. The third method was based on the fact that phosphorylation-induced 45Ca2+ occlusion occurred faster than 45Ca2+ dissociation from nonphosphorylated ATPase: here, we measured the rate of 45Ca2+ internalization on addition to 45Ca(2+)-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two 45Ca2+ ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the non-phosphorylated state of the ATPase. We found that after ATP-induced phosphorylation, the two 45Ca2+ ions dissociated toward the lumenal medium with virtually identical rate constants; this was observed under different ionic and pH conditions and also in the presence of a high Ca2+ concentration. As a control, the same partial isotopic exchange procedure allowed us to confirm that, in contrast, when ATP was absent from the final dissociation medium, the two 45Ca2+ ions dissociated from nonphosphorylated ATPase toward the cytoplasmic medium at different rates, the one bound more deeply only dissociating after a lag period corresponding to dissociation of the superficial one.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium measurements under the same conditions. These results are consistent with sequential dissociation of calcium from a narrow binding pocket inside which a single calcium ion can move fairly easily. Escape of calcium might be controlled by a structural compartment acting as a gate.

Adenosine Triphosphate↗
PubMed1991

Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses.

In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses.

Animals↗
PubMed1991

Electrochemotherapy of spontaneous mammary tumours in mice.

Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours.

Animals↗
PubMed1991

[Electrochemotherapy, a new antitumor treatment: first clinical trial].

Electrochemotherapy (ECT) is a new antitumor treatment which consists in delivering electric pulses to the tumor some minutes after an intravenous injection of bleomycin. We report here the first clinical trial of ECT, applied to patients with permeation nodules of head and neck squamous carcinomas. ECT was well tolerated by patients, no serious incident occurred and a clear antitumor efficiency was found.

Bleomycin↗
PubMed1990

Effects of phospholipids on binding of calcium to (Ca2(+)-Mg2(+)-ATPase.

The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.

Androstenols↗
PubMed1990

Mechanism of inhibition of the (Ca2(+)-Mg2+)-ATPase by nonylphenol.

The effects of nonylphenol and 3,5-dibutyl-4-hydroxytoluene (BHT) on the activity of the (Ca2(+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum have been studied. At high concentrations, both inhibit the ATPase activity of the ATPase either in native lipid or in bilayers of dioleoylphosphatidylcholine but, at low concentrations, an increase in ATPase activity is observed, particularly for the ATPase reconstituted into dimyristoleoylphosphatidylcholine. Neither nonylphenol nor BHT binds at the lipid-protein interface of the ATPase. Nonylphenol decreases the effective equilibrium constant for phosphorylation of the ATPase by Pi probably through an increase in the effective rate of dephosphorylation of the phosphorylated ATPase. It also decreases the effective rate of the E2-Ca2E1 transition and increases the effective equilibrium constant E2/E1 for the ATPase. Inhibition of ATPase activity follows from the slowing of the E2-E1 transition despite increases in effective rates for dephosphorylation and for the transport step, Ca2E1P-E2P. Since nonylphenol has been shown to affect equilibrium constants for various steps in the reaction pathway of the ATPase, inhibition of activity of the ATPase cannot follow from effects on the fluidity (viscosity) of the membrane, since fluidity alone cannot affect equilibrium properties of the system.

Animals↗