Inhibitory effects of the combination of HER-2 antisense oligonucleotide and chemotherapeutic agents used for the treatment of human breast cancer.

Poor response to chemotherapy in patients with breast cancer is often associated with overexpression of HER-2/neu. Interference with HER-2 mRNA translation by means of antisense oligonucleotides might improve the efficacy of chemotherapy. To test this hypothesis, eight breast cancer cell lines and a normal human fibroblast cell line were examined for their level of HER-2 expression, their sensitivity to phosphorothioate antisense oligonucleotides (AS HER-2 ODN), and to various chemotherapeutic agents, and the combination of the two. No correlation was found between the intrinsic HER-2 level and either the sensitivity to a particular chemotherapeutic agent alone, or the amount of growth inhibition observed with a specific AS HER-2 ODN concentration. Although sequence specificity and extent of AS HER-2 ODN inhibition of HER-2 synthesis were somewhat higher in the HER-2 overexpressing MDA-MB-453 and SK-BR-3 cells, we found that antisense treatment significantly sensitized all of the breast cancer cells, even MDA-MB-231 and MDA-MB-435 cells, with approximately basal levels of HER-2, to various chemotherapeutic agents. In addition, the combination of AS HER-2 ODN and taxol was shown to synergistically induce apoptosis in MDA-MB-435. These results demonstrate that overexpression of HER-2 would not be a prerequisite for the effective use of AS HER-2 ODN as a combination treatment modality for breast cancer and suggest that the use of AS HER-2 ODN, as part of a combination treatment modality, need not be limited to breast tumors that display elevated levels of HER-2.

[Diastereomers of nonionic analogs of nucleic acids. V. Alkylation of nucleic acids in the living cells by ethylated derivatives of oligonucleotides containing the residue of nitrous yperite. The effect of the phosphotriester fragment configuration]. / Issledovnie diasteromerov neionnykh analogov oligonukleotidov. V. Alkilirovanie nukleinovykh kislot v zhivykh kletkakh étilirovannymi proizvodnymi oligonukleotidov, soderzhashchikh ostatok azotistogo iprita. Vliianie konfiguratsii fosfotriéfirnogo fragmenta.

The alkylation of the cell biopolymers (RNA, DNA, proteins) by reagents Tp'(Et)Tp'(Et)Tp'(Et)TpU(CHRCl) (1) Tp'(Et)Tp''(Et)Tp'(Et)TpU(CHRCl) (2) Tp''(Et)Tp'(Et)Tp''(Et)TpU(CHRCl) (3) Tp''(Et)Tp''(Et)Tp''(Et)TpU(CHRCl) (4) Tp(Et)Tp(Et)Tp(Et)TpU(CHRCl) (5) Tp(Et)Tp(Et)Tp(Et)Tp(Et)U(CHRCl) (6) TpTpTpTpU(CHRCl) (7) where (CHRCl) is the residue of 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]-benzylidene has been investigated in the case of the ascite carcinoma Krebs-2. p' and p" designate the enantiomeric configurations at the internucleotide phosphorus atoms of the triester fragment--Tp(Et)T--, and p designates the racemic mixture. Completely and partly ethylated reagents (1)-(6) have been found to bind to the cells 4-15 fold more effectively than the diester derivative. The concentration of reagents (1)-(6) in the cells is 2-7 fold higher than in the external medium. Among the diastereomers (1)-(4) reagent (4) with the p"-configuration is the most efficient in binding with the cells 2-3 fold more efficient than reagents (1)-(3). The main targents of modifications performed in the cells by means of reagents (1)-(7) have been established. These are RNA, DNA and proteins. The share of the reagents which react with nucleic acids increases from 45% [reagent (1)] to 80% [reagent (4)], and that reacting with proteins decreases from 50 to 20% correspondingly. Reagent (4) with the p" configurations at phosphotriester fragments alkylates nucleic acids most effectively among the phosphotriester diastereomers (1)-(4): 11-fold more efficient than reagent (1) with configuration p'. The extent of modification of poly(A)+-tracts of m-RNA by reagent (4) in comparison with reagent (1) is 50-fold higher.

[Selective modification of polyadenyl fragments of mRNA from Krebs-2 ascites carcinoma cells by an alkylating derivative of nonathymidilyluridine]. / Napravlennaia modifikatsiia poliadenilovykh fragmentov informatsionnoi RNK v kletkakh astsitnoi kartsinomy Krebs-2 alkiliruiushchim proizvodnym nonatimidililuridina.

It is shown that alkylating reagent 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]-benzylidene nonathymidilyluridine penetrates into the Krebs-2 ascite carcinoma cells and efficiently alkylates their polymers. Nearly 30% of the reagent penetrated into the cell is consumed by nucleic acids. In conditions providing stability of the complementary complexes the modification extent of poly(A) fragments is two orders of magnitude greater than that of other nucleic acids fractions. No destruction of the oligonucleotide moiety of the reagent occurs in the course of intracellular alkylation.

[Interaction of alkylating oligonucleotide derivatives with mammalian cells. A study of the uptake mechanism of derivative cells]. / Vzaimodeistvie alkiliruiushchikh proizvodnykh oligonukleotidov tkami mlekopitaiushchikh. Issledovanie mekhanizmov zakhvata proizvodnykh kletkami.

Interaction of alkylating deoxyribooligonucleotide derivatives, bearing 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residues at their 5'-terminal phosphates, with mouse fibroblasts L929 and with ascite carcinoma cells Krebs 2 has been investigated. It was found, that the derivatives are taken up by the cells according to the endocytosis mechanism. At high concentration of the oligonucleotide derivatives in the cultivation medium (greater than 10 microM), the fluid phase endocytosis is the major pathway of uptake; binding of the derivatives by the cells is partially reversible and their intracellular mean concentration approaches 1/20 of their extracellular concentration. At low concentration of the oligonucleotide derivatives, the predominant mechanism is the more efficient adsorption endocytosis; at concentration of the derivatives less than 0.5 microM, their mean intracellular concentration exceeds that in the culture medium. Stability of the oligonucleotide derivatives in cells depends on their nucleotide composition. Their nucleolytic degradation rate is low enough to allow them to react with cellular biopolymers.

[Synthesis, properties and interaction with eukaryotic cells of alkylating derivatives of oligodeoxyribonucleotides containing cholesterol or phenazinium residue covalently bound to the 3'-terminus]. / Sintez, svoistva i vzaimodeistvie s éukarioticheskimi kletkami alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov, soderzhashchikh kovalentno prisoedinennyi k 3'-kontsu ostatok kholesterina ili fenaziniia.

Radioactive alkylating 5'-[32P]-[4-(N-2-chlorethyl)N-methylaminobenzyl]-5'-phospham ide decadeoxyribothymidilate derivatives containing either free hydroxyl group (reagent I), hydrophobic cholesterol residue (reagent II) or polyaromatic phenazinium residue (reagent III) at 3'-termini were synthesized. The products were purified by HPLC and used for oligonucleotide-directed alkylating of DNA in isolated rat liver nuclei, Krebs-2 ascite carcinoma cells and L-929 murine fibroblasts. The uptake of reagent II by the cells was two orders of magnitude higher than that of reagent I and III. Intracellular alkylation of DNA by reagent II both in isolated nuclei and in living cells was about one order of magnitude higher than in the case of reagent I. The presence of phenazinium at 3'-termini of the reagent III leads to a sufficient increase of the alkylation extent compared to reagent I despite a quite low extent of its uptake by the cells.

[The effect of modifying terminal oligonucleotide linkages on their stability in cell cultures]. / Vliianie modifikatsii kontsevykh zven'ev oligonukleotidov na ikh stabil'nost' v kul'ture kletok.

The effect of modification of terminal groups of deoxyribooligonucleotides on their stability in cell culture and inside mammalian cells, namely Krebs 2, ascite carcinoma (KAC) and mouse fibroblasts L929, has been investigated. Oligonucleotides and their derivatives were found to be stable in culture medium without serum during 24 h. In the medium with KAC cells or ascitic fluid, orthophosphate was rapidly eliminated from the 5'-terminus of the oligonucleotides. In KAC cells, the scission of 5'-phosphomonoester bonds was accompanied by reutilization of the phosphate and by degradation of oligonucleotides to mononucleotides. In the medium with fibroblasts L929, the oligonucleotides were degraded from the 3'-end to tetranucleotides. Modification of oligonucleotides at the 5'-terminus by amidation made the 5'-phosphate groups resistant to KAC. Modification of the oligonucleotides by coupling of cholesterol or phenazinium to the 3'-terminus sufficiently increases their stability in the medium with fibroblasts L929, in that with Krebs 2 ascite carcinoma cells and inside the cells.

[Infection by influenza virus induces production of nucleases by fibroblasts]. / Infitsirovanie virusom grippa indutsiruet produktsiiu nukleaz fibroblastami.

The stability of oligodeoxyribonucleotides and activity of DNAses and RNAses in chicken fibroblasts and in the cells infected by influenza virus have been investigated. It was found that viral infection results in an increase of nucleolytic activity in cells. The fact should be taken into account when planning experiments with antisense oligonucleotides and virus infected cells.

[Chemical transformation of radioactive 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides during in vivo experiments]. / Khimicheskie prevrashcheniia radioaktivnykh 4-(N-2-khlorétil-N-metilamino)benzil-5'-[32P]fosfamidov oligodezoksiribonukleotidov pri postanovke éksperimentov in vivo.

The practical use of reactive oligonucleotide derivatives for complementarily addressed modification of nucleic acids in vivo includes several steps, at which side chemical reactions resulting in a decrease of the modification efficiency may take place. Chemical reactions of 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides were studied in vivo. The intermolecular self-alkylation at the reactive residue of the alkylating derivative was found in the precipitate of its lithium salt under acetone at-20 degrees C. The effects of pH, buffer solutions, salts, temperature, phenol, cell culture suspensions, tissue homogenates, etc., on the stability of the derivatives were studied. A sufficient cleavage of the phosphamide bond was observed at pH less than 3. In fresh liver homogenates the nucleolytic degradation of the oligonucleotide part of the reagent was shown to occur. After intraperitoneal injection of mice with radioactive alkylating derivatives up to 50% of the reagent was included into the blood biopolymers within one hour. The covalently linked to the biopolymers oligonucleotide appeared to be highly degraded thereby.

[The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]. / Issledovanie stabil'nosti alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov, soderzhashchikh prisoedinennyi k 3'-kontsu ostatok kholesterina ili fenaziniia, pri vzaimodeistvii ikh s kletkami Acholeplasma laidlawii PG-8.

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.

[The interaction of alkylating derivatives of oligodeoxyribonucleotides and their methylphosphonate analogs with Mycoplasma cells]. / Vzaimodeistvie alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov i ikh metilfosfonatnykh analogov s kletkami mikoplazm.

Alkylating derivatives of decathymidylates and methylphosphonate analogs of oligodeoxyribonucleotides (MPAO) were studied for their interaction with cells of Acholeplasma laidlawii PG-8, Mycoplasma capricolum California Kid, M. pneumoniae FH and phytopathogenic strain (St. 118). It is shown that MPAO of octa- and hexadecathymidylates as well as decathymidylates 3'-terminal modified by phenazine and cholesterol groupings are sorbed by mycoplasma cells and can penetrate inside the cells. Efficiency of binding of alkylating derivatives and MPAO with mycoplasma cells depends on interaction time of reagents, their concentration in the reaction mixture and temperature.