Biomedical subjects

E Gendaszewska

Publications and source records attributed to E Gendaszewska.

5 recordsLinked to original sources

PubMed2001

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5'-nucleotidase.

Many reports indicate different nonantisense yet sequence-specific effects of antisense phosphorothioate oligonucleotides. Products of enzymatic degradation of the oligonucleotides can also influence cell proliferation. The cytotoxic effects of deoxyribonucleoside-5'-phosphates (dNMPs) and their 5'-phosphorothioate analogs, deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) on 4 human cell types (HeLa, HL-60, K-562, and endothelial cells) were examined, and the effects were correlated with the catabolism of these compounds. The results indicate that differences in cytotoxicity of dNMPs or dNMPSs in these cells depend upon different activity of an ecto-5'-nucleotidase. It has also been found that dNMPSs stimulate proliferation of human umbilical vein endothelial cells and HL-60 cells in a concentration-dependent manner. This stimulation might be caused by the binding of deoxynucleoside-5'-phosphorothioates to as-yet unidentified nucleotide receptor(s) at the cell surface. (Blood. 2001;98:995-1002)

5'-Nucleotidase↗

PubMed2001

Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma.

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.

Animals↗

PubMed2000

Multidrug resistance-associated protein--reduction of expression in human leukaemia cells by antisense phosphorothioate olignucleotides.

Multidrug resistance-associated protein (MRP1) causes cellular drug resistance in several cancer cell lines. In this paper we show that antisense oligonucleotides decrease MRP1 expression in human leukaemia cells. We investigated biological activity of a series of 12 linear phosphorothioate oligonucleotides, complementary to several regions of MRP1 mRNA. The oligonucleotides were administered to leukaemia HL60/ADR cells overexpressing MRP1 protein. Then, the level of MRP1 mRNA was determined by means of semiquantitative RT-PCR and the protein level by reaction with specific monoclonal antibodies. Some of the investigated antisense oligonucleotides decrease the expression level of the MRP1 protein by 46% and its mRNA level by 76%.

DNA-Binding Proteins↗

PubMed1999

Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.

Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity. For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.

Endodeoxyribonucleases↗

PubMed1998

[DNA vaccines].

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Autoimmune Diseases↗