Expression of human growth hormone in cultured mouse fibroblasts

The new system for the transfer and expression of foreign genes based on retroviral vectors pPS-neo, conferring neomycin resistance was constructed. The BALB/c mouse cell lines producing highly active human growth hormone (more than 7 micrograms/ml into culture medium) were constructed using these vectors. An antibody column was used to purify the growth hormone from cell culture medium. Possibilities of producers to be applied for gene therapy are discussed.     

Identification of a protein product of a novel human gene SON and the biological effect upon administering a changed form of this gene into mammalian cells

We have investigated the functional activity of human son gene, that possesses the homology to mos and myc genes. Specific antibodies (antiserum) were raised to synthetic peptide, that corresponds to son-protein 943-963 amino acid residues. With this antiserum the presence of son-protein was showed in lysates of cultured human cells transformed by adenovirus type 5, RAT 2 cells and primary human embryonic fibroblasts. son-Protein molecular weight (92 kDa) was determined by the method of electrophoresis in SDS-polyacrylamide gel. Thus, it was shown the presence of son gene protein in animal and human cells. To determine a possible son gene role in mammalian cells we have cloned the 3' part (2667 b.p.) of son cDNA in retroviral vector pPS-3-neo. Transformed cells of different lines were selected. A large portion of this cells changed their morphology. New protein product (120 k), that reacted with antiserum to son specific peptide, was found together with p92son in these clones.     

Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.     

Improved efficient gene transfer into insect and mammalian cells by baculovirus vectors

The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.     

Binase possesses a selective cytotoxic action on kit-transformed precursors of myeloid cells

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.     

Modulation of activated oncogene c-kit expression with RNA-interference

Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells as well as Kasumi-1 cells from the patient with AML.     

The control of DNA replication in hybrids between neutrophils and fibroblasts

Abstract. DNA synthesis regulation in heterokaryons between mouse neutrophils and cultured cells of various proliferative potentials has been studied. The following features have been found. Both immortalized and non-immortalized cells can reactivate DNA synthesis in neutrophil nuclei. The reactivation ability of cultured cells increases after immortalization and is not changed by further transformation. Neutrophils inhibit the entry of cultured cell nuclei into S phase and have no effect on ongoing DNA synthesis. Malignant cells are much less sensitive to the inhibitory action of neutrophils than non-malignant ones. Non-malignant immortalized cells are as sensitive to this effect as non-immortalized cells. Neutrophil karyoplasts do not influence DNA synthesis in partner cultured cell nuclei. Cycloheximide pretreatment of neutrophils drastically diminishes their inhibitory effect.     

Human RFP2 gene promoter: unique structure and unusual strength

Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.