Biomedical subjects
Publications and source records attributed to L M Mir.
Here, we present a rapid and reproducible procedure based on square-wave pulse electroporation that allows efficient penetration of synthetic oligonucleotides into intact yeast cells. This procedure was successfully used to modify the yeast genome with small amounts of oligonucleotide.
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P-glycoprotein (P-gp), a plasma membrane glycoprotein associated with the multidrug resistance phenotype, is responsible for the ATP-dependent efflux of various amphiphilic drugs. Using membrane vesicles prepared from the multidrug resistant cell line DC-3F/ADX, we studied the perturbation of the basal (i.e. in the absence of drug) and verapamil-dependent P-gp ATPase activities induced by various detergents, at non-solubilizing, as well as at solubilizing, concentrations. The progressive membrane solubilization with increasing detergent concentration was monitored by light scattering and centrifugation experiments. For non-solubilizing detergent concentrations, all tested detergents except DOC induced a partial inhibition of P-gp ATPase activity, which was not correlated with the amount of the various tested detergents incorporated in the membranes. Analysis of the verapamil-induced P-gp activation reveals that P-gp ATPase activity is differently modulated by the various detergents at non-solubilizing concentrations. Thus, specific interactions between P-gp and detergents are more likely to occur rather than a global membrane perturbation. After solubilization by the various tested detergents, the basal P-gp ATPase activity was virtually completely inhibited, except in the presence of CHAPS which was able to preserve this activity at a level comparable to that measured in native membranes. However, the verapamil-induced P-gp ATPase activation was lost during P-gp solubilization by CHAPS, but recovered after dilution of CHAPS below its critical micellar concentration. These observations indicate specific interactions between P-gp and CHAPS molecules within the mixed micelles. On the whole, our data evidencing specific interactions P-gp/detergents are consistent with the location of the drug transport sites on P-gp transmembrane domains.
P-glycoprotein, a plasma membrane protein overexpressed in multidrug-resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy-dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR-reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P-glycoprotein interaction with various compounds, we measured the P-glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC-3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P-glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half-maximal activating concentrations of approximately 1.5 microM and approximately 25 microM, and activation factors of approximately 1.7 and approximately 2.2. Vinblastine inhibits the activation of P-glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant approximately 0.5 microM in both cases. This demonstrates that vinblastine has a specific modulating site on P-glycoprotein. The combined modulation of P-glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P-glycoprotein ATPase activity and on the MgATP concentration dependence of P-glycoprotein ATPase activity, they could bind P-glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P-glycoprotein ATPase by vinblastine and progesterone reveals a non-competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P-glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P-glycoprotein ATPase stimulation in a non-competitive manner, these two molecules can also bind independently P-glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P-glycoprotein ATPase. Three MDR-related molecules, taken as models for interaction with P-glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P-glycoprotein function.
Electrochemotherapy combines bleomycin and local electric pulses that allow cell permeabilization and free access of bleomycin to its intracellular target. We report the first veterinarian clinical trial of electrochemotherapy in 12 cats with spontaneous large soft-tissue sarcomas that suffered relapse after treatment with conventional therapies. Permeabilizing electric pulses were delivered using external surface electrodes, as well as new needle-shaped electrodes that were designed to be inserted in tumours for more effective treatment of several-centimetre-thick tumour nodules. The electric pulses were applied to the tumours several times from 4 to 15-30 min after a bolus intravenous injection of 0.5 mg kg(-1) bleomycin. Tolerance to treatment was excellent without general side-effects. The cats showed local inflammatory reactions for a few days and disease stabilization lasted from 2 weeks to 7 months. One partial regression was observed, and the general absence of nodule volume decrease can be explained by local fibrotic reactions. Histological analysis of biopsies also revealed massive tumour cell death. The cats' lifespan increased (P<<0.001), with a mean survival time of 6.1 months (maximum 18 months) compared with 0.8 months (maximum 1.5 months) for a group of 11 untreated control cats displaying similar carcinological features. Electrochemotherapy is clearly effective as a salvage treatment for large spontaneous solid tumours in adverse clinical situations and this is promising for future applications.
Electrotherapy by low level direct current has been demonstrated to have antitumor effects in different murine tumor models and in clinics. Electrotherapy in "field" configuration, where electrodes are placed subcutaneously outside of the tumor in a way that tumor lies in between the electrodes, was performed in immunodeficient nude and immunocompetent mice. Electrotherapy was much more effective in immunocompetent mice based on the observed tumor growth retardation, thus demonstrating that antitumor effectiveness of electrotherapy greatly depends on host's immune response. Further experiments were conducted by combining electrotherapy with concomitant immunotherapy in order to potentiate the antitumor effect of electrotherapy. Immunotherapy consisted of local delivery of genetically engineered cells selected for IL-2 secretion. This combined treatment was much more effective than any of the treatments alone.
P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence of P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20-25 microM and 40-50 microM, and activation factors of 2.3 (at 100 microM progesterone) and 1.8 (at 170 microM desoxycorticosterone). Hydrocortisone above 100 microM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected.
BACKGROUND: Electrochemotherapy (ECT) is a new antitumor approach that combines systemic bleomycin (BLM) with electric pulses (EP) delivered locally at the tumor site. These EP permeabilize the cells in the tissue, allow BLM delivery inside the cells, and increase BLM cytotoxicity. As an extension of our initial Phase I trial on patients with head and neck squamous cell carcinoma (HNSCC) permeation nodules, we tested variations of ECT protocol to determine how to improve it. METHODS: Seven patients with multiple and/or large permeation nodules of HNSCC or of salivary or breast adenocarcinoma were treated in 10 sessions. They received BLM followed by runs of four or eight short (100 microseconds) and intense (1000 or 1300 V/cm-1) EP delivered at adjacent positions on the nodules to cover all of the tumor surface. RESULTS: We determined the therapeutic window for EP delivery to be between 8 and 28 minutes after BLM intravenous injection. We showed patient tolerance to a high number of EP, along with ECT feasibility after BLM intraarterial injection or on adenocarcinoma nodules. Clear antitumor effects were obtained, especially in the small nodules. In the largest nodules we observed extended tumor necrosis. CONCLUSIONS: Relatively efficient ECT can be performed for large and think nodules, and ECT remains safe even when a large number of EP are delivered. However, in this study, ECT's effectiveness on large nodules was lower than on the previously treated small nodules, probably due to external electrodes inadequacy. The data reported stimulated us to design a new device for EP delivery.
BACKGROUND: Electroporation is a process that causes a transient increase in the permeability of cell membranes. It can be used to increase the intracellular concentration of chemotherapeutic agents in tumor cells (electrochemotherapy; ECT). A clinical study was initiated to determine if this mode of treatment would be effective against certain primary and metastatic cutaneous malignancies. A group of six patients, three with malignant melanoma, two with basal cell carcinoma, and one with metastatic adenocarcinoma, were enrolled in the study. the treatment was administered in a two-step process. METHODS: Each patient received a 10 unit/m2 dose of bleomycin administered intravenously at 1 to 1.5 units/minute. This was followed by eight 99 microsecond pulses at an amplitude of 1.3 kV/cm administered directly to the tumors 5 to 15 minutes after the bleomycin was completely infused. Pulses were administered after the injection of 1% lidocaine solution around the treatment site. RESULTS: Two of three melanoma patients had objective responses. In these two patients, five of six treated tumors decreased in size, and three completely responded. Untreated tumors displayed continued growth. Objective responses were observed in both basal cell carcinoma (BCC) patients. One patient had partial responses in both treated tumors. The other patient had one of four primary BCCs respond completely, and the remaining three respond partially. Patients with metastatic breast adenocarcinoma showed complete responses in both treated nodules after ECT. All patients tolerated the treatment well with no residual effects from the electric pulses. CONCLUSIONS: ECT was an effective local treatment in the majority of nodules treated. The results thus far are very encouraging and the study is being continued.
1. Bleomycin (BLM) is a cytotoxic drug currently used in anticancer chemotherapy. We present here a summary of the well established "old" knowledge concerning BLM structure, properties and activity at the molecular level, as well as its current clinical uses. 2. Then, we introduce "new" recent facts concerning BLM interaction with animal cells, that demonstrate the peculiar characteristics of that drug and its potentialities. Indeed, BLM has an high intrinsic cytotoxicity (i.e. it is a very potent drug once inside the cell). However, BLM cytotoxicity is limited because BLM is unable to diffuse through the plasma membrane. The very low amounts of BLM that can reach the cell interior enter the cells by a mechanism that requires BLM interaction with a plasma membrane protein. 3. Finally, we present and discuss a new possible use of BLM in an antitumor approach that we term electrochemotherapy. This new treatment, still under development, is based on the increased BLM delivery into tumor cells after cell permeabilization by electric pulses administered locally at the solid tumor site. This results in a huge local potentiation of BLM activity, in the absence of systemic adverse effects. Clinical trials are in progress.
The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G0/G1 cell cycle region (the sub-G1 region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G1 region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies.
Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2-producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2-secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers.
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.
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.
Accumulation of boron in tumor tissue is an indispensable requirement for boron neutron capture therapy and it is important that the uptake is as high as possible. In this work we have studied the influence of electropermeabilization in vivo on the uptake of boron in normal and RG 2 glioma bearing Fischer 344 rats. Two different boron compounds, a sulfhydryl boron hydride (BSH) and a boronated porphyrin (BOPP), have been investigated. The rats were infused intravenously during 5 min with 175 micrograms BSH/g body weight or 12 micrograms BOPP/g body weight. Two electrodes were placed 5 mm apart in the brain and electropermeabilization was performed with eight square 400 V pulses at 4 and 7 min after the end of the infusion. After 6 h the animals were killed, and the boron content in the tumors and the surrounding brain was measured with neutron-activated autoradiography. In electropermeabilized healthy animals the BOPP uptake was low and limited to the electrode lesions, whereas BSH was spread extensively throughout the hemisphere. Rats with gliomas showed doubled (BOPP) to 10-fold (BSH) uptake of boron in the tumor when electropermeabilization was performed as compared with untreated animals. We conclude that electropermeabilization in the future may provide an interesting possibility to increase the uptake of certain boron compounds before neutron capture therapy.
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We summarize the way which, starting at the electropermeabilization of cultured cells, a technique currently used in molecular and cellular biology, had led up to the electrochemotherapy, the new antitumor treatment that we have designed at the Institut Gustave-Roussy. Electrochemotherapy consists in intravenous administration of a low dose of bleomycin followed by local delivery of electric pulses on tumor nodules by means of two electrodes located at each side of the nodule. We describe the basis of the electrochemotherapy and we report the results obtained in mice and in clinical trials. Lastly we discuss the reasons for which electrochemotherapy, a treatment relevant to some precise clinical situations, might be widely instrumental in the treatment of cancer.
Bleomycin (BLM), a currently used anticancer drug, crosses the plasma membrane poorly. Electropermeabilization allows a defined number of BLM molecules to enter directly into the cell cytoplasm. Such a procedure has revealed that BLM is endowed with a very high intrinsic cytotoxicity. Here we show that when several million molecules of BLM are internalized, morphological changes identical to those usually associated with apoptosis are observed as well as very rapid DNA fragmentation into oligonucleosomal-sized fragments. We demonstrate that this fragmentation, which occurs within a few seconds after BLM internalization, is consistent with a direct internucleosomal cleavage of chromatin by BLM. This result reinforces the importance of DNA digestion as an early and essential step in the morphological changes associated with apoptosis. Our study also demonstrates that BLM cytotoxicity is actually due to the DNA double strand breaks generated by this drug.