[Apoptosis and neutrophils on the regulation of proliferation and differentiation ex vifo of myeloid cells with Ph-chromosome].

Human myeloid cells with Ph chromosome (Ph+ cells) from chronic myeloid leukemia (CML) in the course of proliferation and differentiation ex vivo are regulated under alternation of cell proliferation and neutrophil maturation stages by consecutive blocking and inducing apoptosis with of neutrophils participation as well bcr/abl, bax and bcl2 genes expression. Apoptosis regulation of three main Ph+ cells types from CML patients depends on alternation sequences of proliferation (1) and maturation (2) cell stages and realized by two ways. The first one is performed by consecutive blocking and inducing apoptosis under 2/1/2 stage alternation.

Gene Expression upon Proliferation and Differentiation of Hematopoietic Cells with Ph Chromosome ex vivo.

The genesp53, mdm2, p21, c-myc,bcr/abl, bcr, bcl2, bax, and gapdh participate in the regulation of cell proliferation and differentiation, apoptosis and cell distribution for the cell cycle ex vivo in the Ph(+)cells of chronic myeloid leukemia containing the Ph chromosome andbcr/abloncogene. Expression of these genes correlates with regulation of cell proliferation and differentiation by alternating proliferation and maturation stages for three main Ph+cell types that occur under chronic myeloid leukemia. Thep53, p21, mdm2, and gapdh genes overexpress in active proliferating myeloid cells in the cell cycle S+ G2/M phases and when the phases are coincident with the proliferation stage.

Cell Regulation of Proliferation and Differentiation ex vivo for Cells Containing Ph Chromosome in Chronic Myeloid Leukemia.

Cell regulation of Ph(+)cell proliferation and differentiation has been studied ex vivo in various chronic myeloid leukemia (CML) patients. The regulation is provided by alternation of effective stages of proliferation and maturation with inhibition of Ph(+) cell proliferation by accumulating neutrophils under apoptosis blockage. The alternation of stages consists of switching stage 1 (effective proliferation) to stage 2 (effective maturation) and proceeds according to the 1/2 -1/2/1 or 2/1-2/1/2/1 schemes.

[Kinetics of proliferation, differentiation and transcription of genes regulating apoptosis in cultured human BCR/ABL+ Ph+-cells].

Ph+, bcr/abl+ cells arise due to t(9,22) chromosome translocation and Ph+ chromosome formation in hematopoietic stem cells. The cells show appreciable apoptosis suppression but retain their ability to differentiate and maturate. Ph chromosome, bcr/abl oncogene and Ph+, bcr/abl+ cells themselves are the hallmark of chronic myeloid leukemia.

[Parameters of binding and transport of oligodeoxynucleotide, which includes BCL2 mRNA translation start, to the K562 cells].

Interaction of oligodeoxynucleotides (ODN), 18-mer, which included sequence of BCL2 mRNA translation start, with K562 cells has been studied. The kinetic curves of interaction showed that oligonucleotide total binding with the cells at 37 degrees C and low oligonucleotide concentration (< or = 30 nM), as well as under lipofection, were composed of two processes: 18-mer surface binding with cell membranes and its non-proportional internalization into the cells. The last, in turn, consisted of three consequent steps.

Binding features of BCL2-targeted oligodeoxynucleotides with K562 cells.

Delivery of various oligodeoxynucleotides into cells is mediated by binding to certain surface proteins followed by receptor-mediated endocytosis. Moreover, oligonucleotides are able to provoke perturbation of cell surface proteins and growth factor receptors among them. Here we described binding sense BCL2 oligodeoxynucleotide targeted to translation start of BCL2 mRNA (ODN) with K562 cells.

[Polymorphism at codon 117 of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene].

A T-to-C substitution, replacing a hydrophobic isoleucine residue with a hydrophilic threonine residue in position 100 of a mature protein molecule, was found at codon 117 of the GM-CSF gene. The mutation frequencies were estimated in 51 DNA samples from healthy adult donors and also in 20 samples from patients with different neoplastic myeloid disorders. Almost equal substitution frequencies in patients and normal individuals were observed, suggesting that the defect was not associated with leukemia.

[Inheritable phenotypic normalization of rodent cells transformed by simian adenovirus SA7 E1 oncogenes by singled-stranded oligonucleotides complementary to a long region of integrated oncogenes].

G11 mouse cells and SH2 rat cells transformed with simian adenovirus SA7 DNA showed inheritable oncogen-specific phenotypic normalization when treated with sense and antisense oligonucleotides complementary to long RNA sequences, plus or minus strands of the integrated adenovirus oncogenes E1A and E1B. Transitory treatment of the cells with the oligonucleotides in the absence of serum was shown to cause the appearance of normalized cell lines with fibroblastlike morphology, slower cell proliferation, and lack of ability to form colonies in soft agar. Proliferative activity and adhesion of the normalized cells that established cell lines were found to depend on the concentration of growth factors in the cultural medium.

[Use of polymerase chain reaction for determining bcr/abl mRNA in human chronic myeloleukemia].

A modified system with two pairs of primers is suggested for the analysis of different bcr/abl mRNA variants, using the polymerase chain reaction (PCR) method. It was demonstrated that, with the use of this method, mRNA preparations obtained from bone marrow cells are more informative than those obtained from blood cells. Fresh preparations of thermostable Tth DNA polymerase, which exhibited the reverse transcriptase activity, appeared to be optimal for PCR analysis of the bcr/abl mRNA pattern.

[DNA probes for the alternative splicing region of the 6th exon of the human CSF-1 gene. Polymerase chain reaction and subcloning].

The DNA probes--pA6-CSF-1 and pA2-CSF-1 for the alternative splicing region of the 6 exon human CSF-1 gene were prepared using PCR and subsequent subcloning in pUC19 plasmid at the XmaI/BamHI sites. Due to the insert sequencing and blotting of human leukocytes DNA, the DNA probes obtained can be useful for screening of mutations in the human CSF-1 gene.

[Mapping of cloned fragments of the macrophage-stimulating factor receptor gene (FMS) from a non-amplified library of human leukocyte genes].

For investigation of FMS gene polymorphism and mutations that reveal functionally meaning in leukemia and myelodysplastic disorders the overlapping recombinants lambda-clones inserted by FMS gene fragments have been obtained from human leukocyte genomic library in the EMBL 3A phage by using oligonucleotide prode (27 nucleotides) based on 12 exon of the FMS gene. 15 DNA probes were prepared by subcloning the lambda-clones obtained in the pBSKS+ plasmid. The probes obtained allow to analyse extracellular, transmembrane and tyrosine kinase regions of the FMS gene independently.

[An evaluation of the prognostic significance of the structure of the chromosomal translocation t (9; 22) locus in chronic myeloleukemia].

The site of the break of chromosome 22 was investigated by blot hybridization and polymerase chain reaction in 41 patients with chronic myeloid leukemia (CML). It is stated that the break site in various regions is not associated with the disease stage and clinicohematological manifestations in the time of the diagnosis. The break in the 5' subregion was indicative of longer survival than that in the subregion 3' patients.

[Cloning the region of chromosome 22 participating in translocation t(9,22) in human chronic myeloid leukemia].

Using oligonucleotide probes, sequences containing the Mbcr locus involved in chromosome translocation t(9:22) were cloned form the library of human genes in the Charon 4A vector. The recombinant clone lambda BCR 1.1 obtained contained Mbcr sequences, but the 3' region of the Mbcr locus in lambda BCR 1.

[Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites].

The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.

[Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines].

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo.

[Penetration of oligo/polynucleotides and their polyalkylating derivatives into rat cells transformed by simian adenovirus DNA].

The transport regularity of the [32P]-oligo/polynucleotides and their polyalkylating derivatives into SH2 rat cells transformed with SA7 adenovirus DNA was investigated. Derivatives penetrate the SH2 cells and their distribution in the subcellular fractions are proportional to the concentration of reagent in the medium. The transport efficiency of the derivatives is inhibited sharply with cell concentration increase and practically does not depend on the action of cell metabolism inhibitors.

Complementary directed modifications of nucleic acids and oncogene-directed mutagenesis in vivo.

High reactivity of the polyalkylating ss oligomers that were sense or antisense 30-200-mers containing sequences complementary to E1 oncogenes of simian adenovirus SA7 and one alkylating residue -CH2CH2N(C2H5OH) (CH2)3N(Ph-p-CH2OH)CH2CH2Cl per each 25 bases of oligomers was demonstrated in vitro by alkylation of ss DNA of recombinant M 13 mp8E1 and mp9E1 phages with inserted E1 sequences of adenovirus oncogene and then by followed complete and selective elimination of E1 sequences from recombinant ss DNA. Treatment of rodent cell cultures transformed by oncogenic SA7 with polyalkylating oligomers which are complementary to the long region of the minus or plus chains of E1 DNA of SA7 revealed a rather high extent of mutant cell clones formation. The cells formed were normalized; they had lost some properties of the transformed cells.

[Detection of carriers of hemophilia A by testing for HindIII polymorphism in the factor VIII gene by PCR].

Representatives of 62 families from Moscow and Leningrad with haemophilia A observed in the pedigree were tested for HindIII polymorphism in the factor VIII gene. The proposed scheme of investigation was based on intron 19 of the FVIII gene amplification by the PCR technique followed by restriction analysis with the inner control of hydrolysis. 207 unrelated X-chromosomes were analysed, the frequency of the incidence of the polymorphic HindIII site in the given population found to be 0.

[Detection of hemophilia A carriers by testing polymorphic Bcl I and HINDIII sites using the PCR method with internal splitting control].

A new variant of the PCR test system is discussed which allows one to detect Bcl I and Hind III polymorphic sites of FVIII gene. It can be used for rapid and effective diagnosis of hemophilia A, especially, in combination with the blot-hybridization technique that detects other polymorphic variants of FVIII gene. The method proposed is highly accurate, reliable and simple.

[Complementary-addressed elimination of E1a sequence of simian adenovirus oncogene SA7 from circular single-stranded DNA of recombinant phage M13].

The G fragment of the simian adenovirus SA7 oncogene corresponding to E1a region was cloned into M13mp8 and M13mp9 phages. Single-stranded DNAs of the recombinant phages thus obtained (mp8G and mp9G) partially digested with DNAse II were used to synthesize polyalkylating derivatives capable of specific hybridisation and subsequent alkylation of complementary G sequences of corresponding phage DNAs. After incubation of complementary alkylated DNA in the presence of lysine, the preselected region (G fragment) was specifically eliminated without damaging vector sequences.

[Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13].

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.