Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

DNA topoisomerases I & II cleavage sites in the type 1 human immunodeficiency virus (HIV-1) DNA promoter region.

Topoisomerase sites were mapped in the 5'-long terminal repeat of HIV-1 DNA by agarose and sequencing gel electrophoresis. Topoisomerase II sites were observed in the absence and presence of teniposide and amsacrine in the transcription initiation region and the TATA box, consistent with a possible role of topoisomerase II in transcription. The NF-kB and Sp1 regions were poorly cleaved. Topoisomerase I sites were relatively unfrequent even in the presence of camptothecin. They were absent in the core promoter and were concentrated in the TAR and the upstream region near the junction with the host DNA.

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.

Improved anti-herpes simplex virus type 1 activity of a phosphodiester antisense oligonucleotide containing a 3'-terminal hairpin-like structure.

We synthesized a series of 20-mer antisense phosphodiester oligonucleotides constituting of a 5'-dodecameric sequence, complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) pre-mRNAs IE4 and IE5, flanked in 3' by octameric sequences adopting hairpin-like structures of different stabilities. The presence of the minihairpins in 3' protected the 20-mer phosphodiester oligonucleotides against serum nuclease degradation, this protection being well correlated to the reported melting temperatures of the minihairpins, and to the gel mobilities of the 20-mer oligonucleotides. While no protection was observed using a linear 8-mer, the addition in 3' of the most stable minihairpin--H8--increased more than eightfold the nuclease resistance of the linear antisense dodecamer. We analyzed the effect of such a protection on the anti-HSV-1 antisense activities of the oligonucleotides. When bearing H8 in 3', the antisense dodecamer was 10 times more active than in the absence of 3'-flanking sequence, while a linear 20-mer control containing the antisense sequence was only 3 times more active. This work provides the basis for a further rational design of phosphodiester antisense oligonucleotides, taking advantage of the specific properties conferred by their conformations.

Dual topoisomerase I and II inhibition by intoplicine (RP-60475), a new antitumor agent in early clinical trials.

The mechanisms of action of intoplicine (RP-60475), a 7H-benzo[e]pyrido[4,3-b]indole derivative that is presently in early clinical trials, have been investigated. Intoplicine induced both topoisomerase I- and II-mediated DNA strand breaks, using purified topoisomerases. The topoisomerase cleavage site patterns induced by intoplicine were unique, relative to those of camptothecin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and other known topoisomerase inhibitors. Both topoisomerase I- and II-induced DNA breaks decreased at drug concentrations higher than 1 microM, which is consistent with the DNA-intercalating activity of intoplicine. DNA damage was investigated in KB cells in culture by using alkaline elution. Intoplicine induced single-strand breaks (SSB) in a bell-shaped manner with respect to drug concentration (maximum frequency at 1 microM approximately 220 rad-equivalents). SSB formation was fast, whereas reversal after drug removal was slow. Similar bell-shaped curves were obtained for DNA double-strand breaks (DSB) and DNA-protein cross-links. SSB and DNA-protein cross-link frequencies were approximately equal, and no protein-free breaks were detectable, indicating the protein concealment of the breaks, as expected for topoisomerase inhibition. Comparison of SSB and DSB frequencies indicated that intoplicine produced a significant amount of SSB not related to DSB, which is consistent with concomitant inhibition of both DNA topoisomerases I and II in cells. Data derived from resistant cell lines indicated that multidrug-resistant cells were cross-resistant to intoplicine but that m-AMSA- and camptothecin-resistant cells were sensitive to intoplicine. Hence, intoplicine might circumvent topoisomerase I-mediated and topoisomerase II-mediated resistance by poisoning both enzymes simultaneously.

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).

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.

[Electrochemotherapy, a new antitumor treatment: first clinical trial]. / L'électrochimiothérapie, un nouveau traitement antitumoral: premier essai clinique.

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.

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.

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.