Human DNA topoisomerase I inhibitory activities of synthetic polyamines: relation to DNA aggregation.

DNA aggregation by polyamines has acquired importance as a prerequisite for the cellular uptake of DNA for gene therapy. Intracellular polyamines are constitutive components of mammalian cells and their availability is critical for cell proliferation. Interference of polyamine biosynthesis by synthetic polyamines leads to cytotoxicity. Optimization of the polyamine structural parameters is necessary to control their DNA aggregation, cytotoxic or enzyme inhibitory activities. We designed two series of tetra- and hexamines and compared their human DNA topoisomerase I (top1) inhibitory effects with the DNA aggregation properties. We show that hexamines are more efficient inhibitors of DNA relaxation by top1 than tetramines and that they suppress the top1-mediated DNA cleavage while tetramines do not. The DNA aggregation abilities within two series of polyamines correlate with the length of their central methylene chain. By contrast, the top1 inhibition within two series does not show the same correlation but demonstrates a threshold inhibitory effect on going from the (CH(2))(12) to the (CH(2))(14) central chain. We show further that the structures of DNA aggregates formed by polyamines with the (CH(2))(10-12) or with the (CH(2))(14-16) chains are very different. The first are a fluid cholesteric-type phases, whereas the second are well-structured aggregates similar to columnar liquid crystals with high packing density of DNA duplexes. The structures of polyamines-induced DNA aggregates are proposed to be crucial for top1 catalysis. The structure-function correlation described here may serve as a guide for rational design of polyamines with desired DNA-aggregation or anti-top1 activities.

Identification of chromosomal loci associated with non-P-glycoprotein-mediated multidrug resistance to topoisomerase II inhibitor in lung adenocarcinoma cell line by comparative genomic hybridization.

In order to identify genomic changes associated with an etoposide resistance acquisition, we used comparative genomic hybridization (CGH) to compare a human lung adenocarcinoma cell line, A549 wild type, and three sublines, A549-VP1-3, exposed to increasing concentrations of the topoisomerase II inhibitor, VP16. R-banding karyotype, fluorescence in situ hybridization (FISH), and Southern blot for the MLL gene were also performed. The CGH analysis showed that the A549-VP3 cell line shared chemoresistance-specific abnormalities (amplification of 11q23-qter, loss of chromosome 17, and deletions of 2p14-pter and 2q23-q24). FISH analysis confirmed the loss of one chromosome 17 in the three resistant sublines and revealed an increased fragmentation of chromosome 2 in more than two segments, depending on the etoposide concentration. FISH with an MLL gene probe showed additional signals of MLL (from three in the A549-WT to seven in the A549-VP3 cell line) translocated onto several other chromosomes. Southern blot indicated an amplification of the MLL gene, dependent on the etoposide concentration, without gene rearrangement. The CGH results are suggestive of loci that could be associated with the acquisition of an etoposide-chemoresistant phenotype. Deletion of the 2p region has already been reported, without any candidate gene being identified. The role of MLL in leukemogenesis has previously been demonstrated, but its role in the development of other tumors or its significance in the chemoresistance process remains to be elucidated.

Multicellular resistance: a paradigm for clinical resistance?

Research on resistance to cancer treatment was mainly focused for 20 years on multidrug resistance (MDR). No useful method of reversing MDR, suitable for clinical use, has yet emerged from this large quantity of work. The reason could be an inadequate evaluation of the target. When grown in spheroids, cancer cells exhibit a phenomenon known as 'multicellular resistance' (MCR). Tumours in patients seem to present the same characteristics. The mechanisms underlying MCR can be classified into two forms: contact resistance and resistance inherent in the spheroid structure. Mechanisms of MCR include: inhibition of apoptosis, high proportion of quiescent cells, modulation of protein expression (including topoisomerases and repair enzymes), potential permeability problems, presence of a hypoxic and necrotic centre and other possible mechanisms that remain to be discovered. A new therapeutic class of drugs is required to overcome MCR. Compounds, which are able to disrupt communication and binding between tumour cells and their microenvironment, seem to be able to circumvent MCR. Interesting results are obtained in vitro and in vivo in mice with specific antibodies or peptides recognised by cell binding proteins. Interestingly, these compounds also appear to be able to inhibit metastasis. Hyaluronidase has already been used with anticancer drugs in patients and was shown to increase drug potency. The explanation given is that it improves drug penetration into spheroids. We now hypothesise that hyaluronidase, in fact, decreases MCR and thus could be the first member of a new therapeutic class.

Selective enhancement of Raman or fluorescence spectra of biomolecules using specifically annealed thick gold films.

Annealing of "thick" metal films deposited onto a smooth dielectric substrate leads to high-order self-organization of metal clusters on the film surface. This work presents the first experimental evidence that the "thick" gold film (TGF) may be specifically annealed to serve as a substrate for surface-enhanced fluorescence or surface-enhanced Raman scattering (SERS) spectroscopy of the same molecule. High-resolved SERS spectra of mitoxantrone (mitox) were recorded on the TGF annealed at 340 degrees C whereas no Raman enhancement but an increase of mitox fluorescence signal were detected on the TGF annealed at 240 degrees C. The mitox fluorescence was further enhanced by deposition of monolayers of pentanethiol or poly-L-lysine on the surface of annealed TGF. The maximal fluorescence enhancement factor per mitox molecule of approximately 50 that was obtained on the annealed TGF covered with poly-L-lysine makes the system promising for applications in immunofluorescence assays and in microspectrofluorescence analysis.

Raman and surface-enhanced Raman scattering spectroscopy of bis-netropsins and their DNA complexes.

The interactions of three bis-netropsins (bis-Nts), which are potent catalytic inhibitors of DNA-binding enzymes, with three double-stranded oligonucleotides (OLIGs), which contain sites of different specific affinities for each bis-Nt, were analyzed. Raman spectroscopy was performed for selective monitoring of modifications of the bis-Nt or the OLIG structure upon bis-Nt-DNA binding, and surface-enhanced Raman scattering spectroscopy (SERS) was an additional tool for topology studies of ligand-DNA complexes. The spectral data showed conformational changes of both partners (bis-Nt and OLIG) upon complexation. Structural variations of bis-Nts appeared to be dependent on a bis-Nt-OLIG binding constant and were found to be small in the specific DNA binding and highest for nonspecific binding of bis-Nt with the corresponding OLIG. The conformational changes of the OLIGs were varied with a bis-Nt-OLIG binding constant in the same manner. The bis-Nts seemed to induce a perturbation in the OLIG's structure, as well as in the positions of their direct binding. These DNA structural modification effects may explain the inhibition of DNA-binding enzymes in the variety of very distinct DNA-enzyme binding sites by bis-Nts reported previously.

Partial restoration of all trans retinoic acid (ATRA) sensitivity by compactin in ATRA-resistant leukemic cells (ATRA-R HL-60).

The resistance to all trans retinoic acid (ATRA) differentiating treatment is a consequence, in most of the cases, of either increased catabolism or down regulation of ATRA uptake. Recently, we have shown that ATRA efficiency to differentiate HL-60 cells was enhanced about 30 times after its incorporation into Low Density Lipoprotein (ATRA-LDL). Here, we attempted to differentiate the ATRA-resistant HL-60 cells by ATRA-LDL at high concentrations up to 10 microM. No significant differentiating effect was observed, although the LDL receptor sites were evidenced in these cells. To increase the number of LDL receptors, the cells were pre-incubated in lipoprotein-deprived serum medium and compactin (2 microM), both ATRA and ATRA-LDL induced gradual increase of cell differentiation (35%+/-1 and 51.5%+/-5 at 10 microM of ATRA and ATRA-LDL respectively). At 2 and 8 microM, the intracellular concentrations of ATRA were respectively three and four times higher when incorporated into LDL. In addition, ATRA-LDL, in the medium, was better protected against degradation than ATRA. The surprising restoration of free ATRA sensitivity after treatment with compactin suggested the implication of new mechanisms unrelated to the LDL-receptor endocytosis but involving the non-sterol pathway.

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

Nuclear transport as an ultimate step of multidrug resistance.

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.

Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs.

The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based expression systems in attempts to overexpress the enzyme for extensive structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT).

Confocal spectral imaging analysis of intracellular interactions of mitoxantrone at different phases of the cell cycle.

It is suggested that the cytotoxicity of anticancer agent mitoxantrone (MITOX) is related to a complex combination of molecular interactions which lead to slowing of S phase traverse and arresting of cells in G2 phase of the cell cycle or even to an apoptosis at high concentration of MITOX. Here intracellular molecular interactions of MITOX were visualised and studied using the confocal spectral imaging technique in synchronised K562 cells. Localisation, quantitative distributions of MITOX in the polar environment, MITOX bound to hydrophobic cellular structures (MITOXphob), nucleic acid-related complexes of MITOX (MITOXNA) and relative distributions of naphthoquinoxaline (NQX) metabolite and intrinsic cellular fluorescence of porphyrins were measured within cytoplasmic and nuclear compartments (chromosomes) of the G2, S, and M cells treated with 10 or 2 microM of MITOX for 1 hour. Colocalisation of MITOX, NQX metabolite and sites of intrinsic cellular fluorescence indicates an accumulation of MITOX within or near mitochondria. One may suppose that due to high concentration MITOX can compete with natural substrates for binding to the enzymes thus affecting the normal functioning of a mitochondria. A remarkable redistribution of MITOX and its complexes occurs in the M cells. In particular, a prominent amount of MITOX is associated with the surface of chromatids but not with the cytoplasmic structures in M cells. At the present time the exact location of the sites of MITOX accumulation in the M cells is not known. It is thought to be some cytoskeleton/microtubule structures associated directly with the chromosomes. Selective labelling of particular cytoskeleton structures and/or proteins in MITOX treated cells is in the progress now and the question will be addressed using the CSI technique.

The effect of 12-alkoxy modification on the in vitro antileukaemic activity of N-methyl 2,3,8,9-tetramethoxybenzo[c]phenanthridinium salts.

Some members of a series of 12-alkyloxy benzo[c]phenanthridines are potent inhibitors of the growth of P388 tumour cells in vitro, with a strong dependence on the nature of the 12-substituent. Analogues with a quaternary nitrogen in the side chain bind strongly to DNA but are less active against the tumour cells. The multi-drug-resistant cell line Pr8/22 shows less sensitivity to the new compounds. K562 Human leukaemia cells undergo differentiation in the presence of the benzo[c]phenanthridine derivatives with a structure-activity relationship which does not correlate well with potency against the P388 cell line.

[Multicellular resistance: another mechanisms of pleiotropic resistance?]. / La résistance multicellulaire: un autre mécanisme de résistance pléiotropique?

Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo. In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells from often a necrotic core. A multicellular resistance appears in spheroids, it is a result of the cell contact to other cells (homogeneous or heterogeneous cells) and/or to the extracellular matrix. The mechanism of this resistance is not known, nevertheless, it can be hypothesised to be linked to the spheroid centre hypoxia, to the quiescence of a large fraction of the cell population and to the apoptosis inhibition due to the cell contact. The "classical" or "unicellular" mechanisms of resistance, as mdr1, MRP, can coexist with the multicellular resistance, but are not responsible for this resistance. The spheroid model of culture is a good opportunity to study a resistance type which looks close to the tumour resistance found in vivo in mice and in patients. A new class of therapeutic molecules appears that can reverse this multicellular resistance, inhibit tumours growth and preclude metastases. The principal mechanism of action of this new pharmacological class appears to be the disruption of the intercellular adhesion forces. Preliminary results obtained with these compounds in patients are promising.

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

Enhancement of all-trans-retinoic acid efficiency in granulocytic differentiation of HL-60 cells by incorporation into low density lipoprotein.

All-trans-retinoic acid (ATRA) has been proven to lead to complete remission of acute promyelocytic leukemia by inducing differentiation into granulocytes except when an acquired resistance occurred. High levels of low density lipoprotein (LDL) receptor in cancer cells suggested the use of ATRA incorporated into LDL. 50% of HL-60 cell differentiation were obtained with 5 nmoles/l of ATRA-LDL compared to 150 moles/l of ATRA. Maximal differentiation (80%) was reached at 25 nmoles/l and 1,000 nmoles/l respectively. This higher efficiency suggests the involvement of LDL receptor in ATRA-LDL internalization and/or the protection of the drug, from eventual catabolism, by LDL particles.

[Differentiating the activity of anticancer drugs: the role of transcription factors]. / Activité différenciante des médicaments anticancéreux: rôle des facteurs de transcription.

The understanding of the mechanisms responsible for the emergence and evolution of cancers has been in constant progress due to advances in molecular biology. Today it allows to conceive therapeutic alternatives to conventional cytotoxic chemotherapy. Among these, differentiation strategy, which aims at reinducing tumour cells towards a normal phenotype, has known a first clinical application with the use of retinoic acid in acute promyelocytic leukemias. Anthracyclines, traditionally employed in cytotoxic chemotherapy, present also a high potential of differentiation. Their mode of action takes place via the activation of transcription factors, which are proteins that are able to modulate the expression of genes by fixing to regulatory sequences of DNA. These observations therefore allow us to foresee a new pharmacology based on transcription factors for the treatment of cancers.

[Detection of metastatic activity of the MCF-7 multidrug resistant cell line cultured in spheroids]. / Mise en évidence d'une activité métastasiante dans la lignée MCF-7 multidrogue résistante cultivée en sphéroïdes.

The occurrence of solid tumors spreading through the body is a major concern for the clinicians. Moreover, in numerous cases, metastases exhibit a multidrug resistant (MDR) pattern. This dual characteristic still remains supported by few biological explanations. The purpose of our study was to compare invasive properties of sensitive and MDR MCF-7 cells. Spheroids were chosen as experimental model since they exhibit a number of characteristics (i.e. tridimensional structure) close to the growth of an in vivo tumor. MDR spheroids formed more compact structures compared to sensitive ones. In every experiment, spheroids made from sensitive cells were more resistant to doxorubicin than the same cells grown as monolayers, a characteristic not observed with MDR cells. On an other hand, a form of multicellular resistance appeared in spheroids of sensitive cells, a fact which was not present in MDR spheroids. Incubation of MDR spheroids in Boyden's chambers put in evidence increased motility and invasive properties through Matrigel which were not present in sensitive MCF-7 cells. Zymograms of culture media and membrane extracts were performed in polyacrylamide gels. Two metalloproteases, progelatinases A et B were detected in culture media conditioned by monolayers and spheroids of both sensitive and resistant cells. In contrast, 2 unidentified serine proteases were detected only in media conditioned by spheroids of both cell types. An intense band of pro-MMP2 was present only in membrane extracts from MDR spheroids. Taken altogether, these results demonstrate that spheroids of MDR cells exhibit a number of properties which could lead to an increased ability to form metastases.

Sequential overexpression of LRP and MRP but not P-gp 170 in VP16-selected A549 adenocarcinoma cells.

Chemoresistance remains the major obstacle to successful therapy of lung cancer. In order to understand drug resistance mechanisms, the expression of three proteins involved in multidrug resistance (P-gp, MRP and LRP) was studied, using the non-small cell lung cancer (NSCLC) A549 cell line. In addition, 3 levels of resistance were obtained by continuous exposure of cells to etoposide (VP16), which led to a 22-fold increase of the resistance index. The wild-type A549 strongly expressed the LRP protein while MRP protein was found at a moderate level. Induction of resistance paralleled an increase of the expression of the mrp gene and a decrease of the lrp gene; the mdr1 gene was not expressed. Taken together, these results indicate that intrinsically resistant NSCLC cells exhibit a complex pattern of MDR proteins, still susceptible to evolve under treatment. Such a fact would have to be considered in clinical situations.

Piperazine derivatives of butyric acid as differentiating agents in human leukemic cells.

Butyric acid is a potent antineoplastic agent with a well-documented differentiation activity on a wide variety of tumor cells. However, its clinical development is strongly limited by its very short metabolic half-life. In this study we report on the in vitro effects of new original piperazine derivatives of butyric acid on the induction of differentiation and the growth inhibition of human erythroleukemia K562 cells and myeloid leukemia HL60 cells. 1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine (HEPB) and [1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine] butyrate (HEPDB) were efficient in acting on the differentiation and proliferation of both cell lines, whereas 1-phenyl 4-(1-oxobutyl)-piperazine (PPB) and 1-(3,4-methylene dioxybenzyl) 4-(1-oxobutyl)-piperazine (POB) acted only on proliferation rates. Such derivatives did not induce significant toxicity in mice. These preliminary results should enable, by the development of new series of piperazine derivatives, a better understanding of the mechanisms of action of butyric acid and its analogues on the coupling of growth and differentiation of neoplastic cells.