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L M Mir

Publications and source records attributed to L M Mir.

162 records · Page 9Linked to original sources
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

Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized.

Bleomycin (BLM), a compound currently used in anticancer therapy, is unable to cross the plasma membrane efficiently. Electropermeabilization allows a defined number of BLM molecules to enter directly into the cell cytoplasm. Such a procedure has revealed that BLM is intrinsicly highly cytotoxic. Here we show that the mechanisms of the cell death caused by BLM are closely related to the number of BLM molecules introduced into the cell cytoplasm. When only a few thousand BLM molecules are internalized, cells display an arrest in the G2-M phase of the cell cycle and become enlarged and polynucleated before dying. These observations parallel the "mitotic death" seen with ionizing radiations. By contrast, 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 the direct internucleosomal cleavage of chromatin by BLM. Our findings reinforce the importance of DNA digestion as an early and essential step in the morphological changes associated with apoptosis.

Animals↗
PubMed1993

A new brain tumour therapy combining bleomycin with in vivo electropermeabilization.

The potentials of in vivo electropermeabilization in combination with bleomycin in brain tumor treatment have been explored. In the brain of normal Fischer 344 rats, 2 electrodes were placed 5 mm apart. Electropermeabilization was performed with 8 to 12 exponential 400 V pulses with a time constant of 325 microseconds. Some animals were given bleomycin i.v., 1mg/kg b.w., 4 minutes before electric pulses delivery. No adverse effects were recorded during the observation of the animals during the following month. The effect of bleomycin and electropermeabilization upon tumour growth was studied in rats with glioma cells (RG2) implanted in the head of the right caudate nucleus. Treatment was given at different time intervals after the implantation of tumor cells and the effect upon survival was studied. Bleomycin alone did not prolong the survival of the animals. On the contrary, bleomycin plus electropermeabilization on the 10th, 11th or 12th day after inoculation increased the survival time to almost double that of untreated animals. We conclude that this treatment may be of value in brain tumour therapy.

Animals↗
PubMed1993

Identification of a plasma membrane protein that specifically binds bleomycin.

In this paper, the association of radiolabelled bleomycin on the plasma membrane of DC-3F cells is shown to be saturable, displaying classical features of a ligand-receptor binding. This association corresponds to the presence of 140,000 to 400,000 binding sites at the surface of the cells with a half-saturating concentration of 5 microM. Moreover, using a gel electrophoresis procedure to separate membrane proteins incubated with radiolabelled bleomycin, we demonstrate the existence of a membrane protein of about 250kDa able to specifically bind bleomycin. This membrane bleomycin-binding protein could play a major part in the association of bleomycin with the cells and in its further internalization, a process that has not been elucidated yet.

Animals↗
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

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.

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

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

Stable [57Co]-bleomycin complex with a very high specific radioactivity for use at very low concentrations.

A method for the preparation of 57Co-labelled bleomycin (BLM) possessing very high specific radioactivity and suitable for use at the nanomolar concentration range is described, and validated using a biological assay. Chelation of BLM with Co(II) results in a very stable complex. However, association does not occur below the micromolar concentration range. A nanomolar [57Co]BLM solution with maximal specific radioactivity can be easily prepared, without handling unreasonable amounts of radioactivity, provided that: equimolar solutions of BLM and [57Co]Cl2 are first mixed at the micromolar concentration range and that the mixture is then diluted a thousand times to reach the nanomolar concentration range.

Animals↗
PubMed1989

Voltage-dependent introduction of a d[alpha]octothymidylate into electropermeabilized cells.

DC-3F cells were submitted to electric square wave pulses in the presence of a d[alpha]octothymidylate 32P labelled in the 5' position. Radioactivity was incorporated in a voltage-dependent manner and reached a maximum for a field intensity of 1300-1400 V.cm-1. Growth curves and parallel cloning efficiency experiments indicated that cell viability was not altered by electric pulses, alone or in the presence of the oligothymidylate, below a field intensity of 1300 V.cm-1. Using affinity chromatography we extracted the incorporated oligonucleotide and showed that it was not degraded during the electropermeabilization experiment time.

Animals↗
PubMed1988

Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs.

The electropermeabilization (EPN) of living cells allows the uptake of non-permeant molecules and can reveal their potential activity on cells without the constraints of the plasma membrane crossing. We decided to compare the cytotoxicity of some anticancer drugs on electropermeabilized (EP) and non-permeabilized (NEP) cultured DC-3F cells exposed to the drugs for a short time. After EPN, the increase in cytotoxicity varies between 1 and more than 700 times, depending on the usual cell uptake pathway of a given drug. The most relevant increase of toxicity was observed with molecules such as netropsin (200-fold) and bleomycin (700-fold) which in ordinary conditions weakly diffuse through the plasma membrane. Only a 3-5-fold increase of the cytotoxicity was observed with lipophilic drugs able to rapidly diffuse through the plasma membrane (actinomycin D, NMHE) both in the case of drug-sensitive and resistant cell strains. This increased toxicity is clearly related to a facilitated uptake because, after electropermeabilization, the effects of melphalan (a drug which enters intact cells via leucine transporters) are not modulated by the external leucine concentration. Thus, EPN enables us to reveal the intrinsic toxicity of hydrophilic molecules which have a limited access to their intracellular targets. We propose that EPN can be used as a novel screening procedure of new cytotoxic molecules which could be modified thereafter in order to facilitate their cellular uptake.

Animals↗
PubMed1988

Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.

The possibility of introducing definite amounts of nonpermeant molecules into electropermeabilized living cells has been approached by quantifying the amounts of Lucifer Yellow (LY; a 457-Da highly fluorescent molecule) and Phytolacca americana (Pokeweed) antiviral protein (PAP; a 30,000-Da ribosome-inactivating protein) retained by the cells after closure of the electric-field-induced transient structures of permeation. Without the electropermeabilization, these two molecules enter the cell only in very small amounts by fluid-phase pinocytosis. Under our experimental conditions, using the NIH 3T3 cells, the intracellular LY concentration can reach a value equivalent to the extracellular concentration and can be regulated by controlling the external concentration. We describe the use of LY in a rapid and efficient test for the determination of the best electrical-shock conditions of other cell lines. After electropermeabilization, PAP is 2 X 10(5) times more cytotoxic. Its toxicity can be detected at external concentrations (10(-11) M) corresponding to less than 10 internalized molecules per electropermeabilized cell. Therefore, after electropermeabilization, the nonpermeant molecules have a direct access to the cytosol and the biological effect of nonpermeant substances can be revealed.

Animals↗
PubMedDate not supplied

Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses.

We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).

Animals↗