[Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference].

In the present study we have applied the siRNA approach for substantial reduction of AML1-ETO and RUNX1 (K83N) expression, which are frequently found in the leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5'-untranslated region of mRNA for the mutant RUNX1 (K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of the oncogenes following the introduction of shRNAs into cells.

[Cholesterol-modified anti-MDR1 small interfering RNA: uptake and biological activity].

Small interfering RNAs (siRNA) are considered to be potent agents for specific gene silencing, but troubles in delivery of siRNA into cells limit their biomedical application. An accumulation of siRNA coupled with cholesterol residue at the 5'-end of the "passenger" strand (chol-siPHK) was investigated in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfectant levels of both unmodified and chol-siRNAs were very low, whereas transfectant substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency for the chol-siRNA being higher than that for the corresponding siRNA. Multiple drug resistance phenotype reversing activity of anti-MDR1-siRNAs targeted to the 557-577 region of MDR1 gene mRNA was investigated in KB-8-5 cancer cells. The chol-siRNA induced cancer cells' death in the presence of vinblastine doses tolerated before more effectively than the conventional siRNA did.

[The CCNB1, HER2, and PKC silencing induced by small interfering RNA decreases a division of different human cancer cells with interfering RNA decreases a different efficiency].

An abnormality in expression of genes encoding proteins responsible for the cell cycle regulation frequently results to a malignant cell transformation and switches the cellular program from differentiation and apoptosis to continuous cell division. To evaluate therapeutic potential resulted from silencing gene expression of key cell circle regulators in different human cancer cells the siRNAs targeted to HER2, protein kinase C (PKC), and cyclin B1 (CCNB1) mRNAs were used. An effective and specific reducing the CCNB1, HER2 or PKC mRNA level was observed through 48 h after the siCycB1, siHER2 or siPKC transfection, respectively. The HER2, PKC, and CCNB1 gene silencing substantially reduced a growth rate of the cell lines, except HL-60, but did not affect the cell death and apoptosis. The best cell division inhibition was induced by the siCycB1 in SK-N-MC cells and by the siPKC in MCF-7 cells. The data obtained suggest the siRNAs selected inhibit the cell division, and the genes investigated may be used as effective targets for curing oncologic diseases.

[Silencing of c-myc gene expression by enzymatically and chemically synthesized siRNAs].

Small interfering RNAs (siRNA) provide a powerful approach for sequence-specific silencing of gene expression. In the present study we investigated inhibition of c-myc gene expression by siRNAs targeted to the sequence 1452-1470 b. in third exon of c-myc mRNA and to homologous regions in second exons of c-myc (697-715 b.) and N-myc (302-320 b.) mRNAs. siRNAs were prepared enzymatically according to the scheme, including dsDNA-templates preparation using Klenow fragment, separate in vitro transcription of each RNA strand with subsequent hybridization and removal of leader sequences by T1 RNase. Investigation of c-myc gene silencing by siRNAs revealed that enzymatically prepared siRNAs induce stronger inhibition of c-myc expression, than siRNA with the same sequence prepared by chemical synthesis. It was found that down-regulation of c-myc gene expression by investigated siRNAs results in efficient inhibition and even complete arrest of carcinoma cell proliferation, moreover, the extend of growth inhibition correlates with the level of siRNA-mediated reduction of c-myc mRNA.

[Copper-catalyzed cleavage of DNA by arenes]. / Med'kataliziruemoe rasshcheplenie DNK arenami.

DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Binding of transcription factors with functionally significant regions of MDR1 gene promoter during its activation]. / Izuchenie sviazyvaniia transkriptsionnykh faktorov s funktsional'no znachimymi raonami promotora gena MDR1 pri ego aktivatsii.

Interaction of transcription factors with functional regions of gene mdr1 promoter has been studied. Binding of transcription factors to double-stranded (ds) oligonucleotides, mimicking four important regulatory regions before and after treatment of KB and K-562 cell lines with doxorubicin, cytarabine, and vinblastine was analysed by gel-shift assay. Gene induction resulted in enhancement of the main complex A(A1) proteins formation on the all four regions in both the cell lines. Inhibition of each complex formation by an excess of non-radiolabelled ds oligonucleotides suggests the possibility of regions interaction during promoter activation. Another indication of interaction of regulatory regions during activation is overlapping minor complexes sets. The results suggest that the four studied regulatory regions are important for mdr1 gene activation. Corresponding oligonucleotides mimicking these regions can be employed as inhibitors of gene transcription that bind specific transcriptional factors.

[Chemical modification of eukaryotic cell chromatin, directed at d(GT)n-repeats of DNA]. / Napravlennaia po d(GT)n-povtoriaiushchimsia uchastkam DNK khimicheskaia modifikatsiia khromatina kletok éukariot.

DNA and proteins of chromatin from eukaryotic cells were specifically modified by an alkylating derivative of pd(AC)6 (complementary to d(GT) repeats of DNA) containing a 4-(N-methyl-N-2-chlorethylamino)benzylamine residue on its 5'-end. It was shown that the efficiency of modification of both DNA and proteins increases in the presence of spermine and spermidine and sharply decreases after preliminary treatment of chromatin by nuclease S1 under conditions of mild cleavage of single-stranded DNA regions. It was suggested that one of the reasons for the presence of unwound d(GT)n stretches in chromatin DNA accessible for interaction with the complementary oligonucleotide is the B-->Z transition. Proteins specifically alkylated within the chromatin, which most likely are located in the regions of local unwinding of DNA, near the repeats, were analyzed.

[Visualization of DNA segments, interacting with reactive oligonucleotide derivatives in interphase nuclei and metaphase chromosomes]. / Vizualizatsiia uchastkov DNK, vzaimodeistvuiushchikh s reaktsionnosposobnymi oligonukleotidov, v sostave interfaznykh iader i metafaznykh khromosom.

Reaction of (pdT)16 derivatives, bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group on its 5' end and biotin on its 3' end with DNA in interphase nuclei and metaphase chromosomes has been investigated by fluorescence and electron microscopy. The result obtained evidence that in interphase nuclei DNA in active chromatin (nucleolus) is the most available for specific modification. In metaphase chromosomes the modified DNA regions are situated on the surface of chromosome.

[Interaction of photoactive oligothymidylate derivatives with HeLa cell chromatin]. / Vzaimodeistvie fotoaktivnykh proizvodnykh oligotimidilata s khromatinom kletok HeLa.

Photoactive derivatives of d(pT)16, bearing arylazide, nitroarylazide and perfluoroarylazide residues, were used for the complementary addressed modification of DNA and proteins in chromatin. As compared with alkylating derivatives, the photoactive compounds possess higher efficiency and specificity, and shorter incubation times which prevents nucleus from degradation. These reagents can therefore be used for identification of proteins located near to particular DNA regions in chromatin.

[Chromatin proteins in rat liver cells, interacting with reactive oligothymidylate derivatives]. / Belki khromatina kletok pecheni krysy, vzaimodeistvuiushchie s reaktsionnosposobnymi proizvodnymi oligotimidilata.

Upon alkylation of the rat liver chromatin with a hexadecadeoxyribothymidylate derivative bearing 4-(N-2-chloroethyl-N-methylamino)benzylamine residue on the 5'-terminal phosphate two nonhistone proteins were modified under conditions of the reagent's forming complementary complexes with poly(A) sequences in DNA. The sequence-specific nature of the reaction is proved by the competition experiments: free oligothymidylate prevented the proteins from alkylation whereas arbitrary oligonucleotide did not. Modification with the reactive oligonucleotide derivatives can be used for the identification of proteins located in the vicinity of the specific open DNA regions in chromatin.