Targeting MDR1 gene: synthesis and cellular study of modified daunomycin-triplex-forming oligonucleotide conjugates able to inhibit gene expression in resistant cell lines.

Reversal of the multidrug-resistant (MDR) phenotype is very important for chemotherapy success. In fact, the expression of the MDR1 gene-encoded P-glycoprotein (P-gp) actively expels antitumor agents such as daunomycin (DNM) out of the cells, resulting in drug resistance. We show that upon conjugation to triplex-forming oligonucleotides, it is possible to address DNM in resistant cells (MCF7-R and NIH-MDR-G185).

Modulation of the efficiency of a siRNA directed against MDR1 expression in MCF7-R cells when combined with a second siRNA.

Effective silencing of MDR1, one of the genes involved in the multidrug resistance phenotype, can be achieved by the use of an efficient siRNA transfected into the doxorubicin-selected MCF7-R human cell line, alone or combined with a moderately efficient siRNA. On the contrary, there is no MDR1 silencing when it is co-transfected with a control siRNA that does not target the human genome. This results from the limited amount of RISC (RNA-Induced Silencing Complex) in human cells, leading to competition between siRNAs.

Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides.

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured.

Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs.

MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. Because of the high toxicity of the inhibitors of P-glycoprotein (P-gp), the protein encoded by MDR1, attention has been focused on selective modulation of the MDR1 gene. Small interfering RNAs (siRNAs) were shown to be powerful tools for such a purpose, even when used at low concentrations (< or =20 nM) in order to avoid sequence nonspecific effects.

The reduction of P-glycoprotein expression by small interfering RNAs is improved in exponentially growing cells.

Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression. Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon. P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density.

Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.

The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am.

Minimally modified phosphodiester antisense oligodeoxyribonucleotide directed against the multidrug resistance gene mdr1.

In the perspective of reversing multidrug resistance through antisense strategy while avoiding non-antisense effects of all-phosphorothioate oligonucleotides which non-specifically bind to proteins, a minimally modified antisense phosphodiester oligodeoxyribonucleotide has been designed against mdr1, one of the multidrug resistance genes. Its stability in lysates prepared from NIH/3T3 cells transfected with the human mdr1 gene has already been demonstrated. Confocal microspectrofluorometry using a fluorescence resonance energy transfer technique allowed its stability inside living cells to be proven.

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids.

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied.

Best minimally modified antisense oligonucleotides according to cell nuclease activity.

Minimally modified oligonucleotides belong to the second-generation antisense class. They are phosphodiester oligonucleotides with a minimum of phosphorothioate linkages in order to be protected against serum and cellular exonucleases and endonucleases. They activate RNase H, have weak interactions with proteins, and have thus a better antisense efficiency.

Factors influencing the oligonucleotides release from O-W submicron cationic emulsions.

We recently described a positively charged O-W emulsion as a delivery system for oligonucleotides (ON) [Teixeira et al., Pharm. Res.

Hybridization kinetics of oligodeoxyribonucleotides with a d(GCGAAGC) hairpin at the 3'-end.

In order to protect them against enzymatic attack of serum in the antisense strategy, oligodeoxyribonucleotides can be protected on their 3'-side by the sequence d(GCGAAGC) which spontaneously forms a hairpin which is known for its extraordinary stability with regard to thermal denaturation or nuclease degradation (I. Hirao, G. Kawai, S.

Opening of the extraordinarily stable mini-hairpin d(GCGAAGC).

For the purposes of the antisense strategy oligodeoxyribonucleotides can be protected against serum and cell nuclease digestion by tagging at their 3'-end with a sequence naturally forming a very stable hairpin, d(GCGAAGC). This nuclease-resistant hairpin is also known for its high thermostability. We demonstrate in this study that attachment of d(GCGAAGC) at the 3'-end of an oligodeoxyribonucleotide does not hinder hybridization of the 5'-part of this oligonucleotide to a complementary DNA strand.

A UV resonant Raman spectroscopic study of the interaction of metallic derivatives of tetrakis(4-N-methylpyridyl)porphine with polynucleotides.

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)2, poly(dA).poly(dT), poly(dG.

Fluorescence resonance energy transfer analysis of the degradation of an oligonucleotide protected by a very stable hairpin.

In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases.

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids.

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane.

Spectral shape modifications of anthracyclines bound to cell nuclei: a microspectrofluorometric study.

Anthracyclines remain today the medications of choice against a wide spectrum of human cancers. Anthracyclines are fluorescent molecules and microfluorimetric methods are often used to determine their cellular distribution. The use of microspectrofluorometric techniques yields additional information because not only the fluorescence intensity but also the spectral modifications of the chromophore can be used to assess the intracellular drug concentration, its localisation and also eventually its metabolisation.

Comparison of DNA sequence selectivity of anthracycline antibiotics and their 3'-hydroxylated analogs.

The sequence selectivity of three anthracyclines and their 3' hydroxylated analogs (in which an OH replaces NH3+ in the daunosamine at neutral pH) was examined in DNase I footprinting experiments on a 158-bp DNA fragment. We found that chemical modification of the daunosamine at C3' has more drastic consequences for sequence selectivity than chemical modification at C4 and C14 of the aglycone moiety. All anthracyclines and hydroxylated derivatives selectively recognize the triplet PyAPy.

Mitomycin C-induced distortions of DNA at minor alkylation sites.

Reductively-activated mitomycin C (MC) presents a high specificity to the 5'-CG site and to a lesser extent the 5'-GG site. However, its affinity is different for each 5'-CG site. This was evidenced by using the 3'-5' exonuclease activity of T4 DNA polymerase on a short DNA fragment exposed to MC, which was gradually activated by several Na2S2O4 additions.

Distortion after monofunctional alkylation by mitomycin C of a dodecamer containing its major binding site.

The structural distortions of the duplex dodecamer d(ATTAACGTTAAT)2 monofunctionally alkylated by mitomycin C have been studied by the use of chemical probes reactivity and resonance Raman spectroscopy. This sequence contains the 5'-ACGT sequence for which mitomycin C was determined to present the best affinity (S. Kumar, R.

Thermodynamic and structural properties of r(ACC) as revealed by ultraviolet electronic absorption, circular dichroism, 1H-NMR spectroscopy and Monte Carlo simulations.

UV absorption, circular dichroism (CD) and 1H NMR, associated with Monte Carlo (MC) molecular structure simulations have been applied to the study of the trinucleoside diphosphate: r(ACC). The MC study which has been conducted as a function of temperature, is based on random variations of the nucleotide conformational angles, i.e.

Interactions of iron-anthracycline complexes with living cells: a microspectrofluorometric study.

The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed.

Evaluation of the structural modifications induced by mitomycin C on nucleic acids.

The interaction of poly(dG-dC).poly(dG-dC) with mitomycin C, an antitumor antibiotic, has been studied by various spectroscopic methods: circular dichroism, Fourier transform infrared resonance Raman scattering and using fluorescence emission of terbium bound to unpaired guanines as local conformation probe. The results allowed us to confirm the lack of long range modification of the DNA secondary structure upon binding.

Accumulation of degradation products of doxorubicin and pirarubicin formed in cell culture medium within sensitive and resistant cells.

Quantitative study of doxorubicin (Adriamycin), pirarubicin (4'-o-tetrahypyranyladriamycin) and daunorubicin in the nucleus of living cells was performed using microspectrofluorometry. As for the cytotoxic assays, drug-sensitive and drug-resistant K562 cells were incubated for 3 days with concentrations of drug ranging from 4 nM to 1 mM. When drug-sensitive cells were incubated with pirarubicin, the spectrum recorded from inside the nucleus was characteristic of anthracycline intercalated between the base pairs in the nucleus.

Ultraviolet resonance Raman marker bands of the right to left helix structure transitions in DNA and polynucleotide model compounds.

The right to left helix structural transition in purine-pyrimidine alternating copolymers has been extensively studied by vibrational spectroscopies, amongst many other experimental approaches. Here, the use of resonance Raman spectroscopy in the ultraviolet region (223-, 257- and 281 nm excitation wavelengths) to monitor such structural changes is reviewed in the light of new results obtained on poly(dA-dC).poly(dG-dT) on one hand, and the previous results obtained on poly(dG-dC)2, poly(dA-dT)2 and natural DNA (Chicken erythrocytes) on the other.

A porphyrin-DNA exciplex: resonance Raman spectra of electronically excited Cu(TMpy-P4) bound to poly(dA-dT).poly(dA-dT).

The resonance Raman spectra of water-soluble porphyrins, Cu(TMpy-P4) and Ni(TMpy-P4), and their mixtures with DNA, Poly(dG-dC).Poly(dG-dC), and Poly(dA-dT).Poly(dA-dT) were measured using 426 nm pulsed laser excitation (and 556 nm for some applications).