Effects of Ukrain on the activities of DNA-nicking enzymes.

We studied the effects of Ukrain, a novel antitumor drug, on the activities of calcium, magnesium-dependent endonuclease (CME) and manganese-dependent endonuclease (MnDE) in rat liver nuclei, the activity of topoisomerase I assessed by pUC19 plasmid relaxation and CME activity in the nuclei of lymphocytes from colon cancer patients. Ukrain was found to exert a dose-dependent inhibiting effect on both CME and MnDE, similar to that exerted by erythropoietin, which was used as a reference preparation. Both Ukrain and erythropoietin also caused dose-dependent inhibition of topoisomerase I activity. The influence of Ukrain on CME activity in the nuclei of the lymphocytes of colon cancer patients was differential, depending on treatment efficacy. The results suggest that DNA-nicking enzymes may be a target of Ukrain and may mediate its antitumor effects.

Induction of apoptosis in cultured Chinese hamster ovary cells by Ukrain and its synergistic action with etoposide.

The induction of apoptosis by Ukrain, a novel antitumor drug, was studied in Chinese hamster ovary (CHO) cells bearing multiple copies of recombinant human erythropoietin gene incorporated into their genome (cell lines CHO-k38 and -k38/12). Ukrain was found to be capable of the in vitro induction of apoptosis in the cell lines studied. The effect was less expressed in cells with type I multiple drug resistance (k38/12). Ukrain acted synergistically with etoposide, i.e., the combined effect of both agents was evident at significantly reduced concentrations. This suggests that pharmacological compositions of the drugs may reduce the effective doses used in chemotherapy and thus significantly diminish its toxic side effects. Ukrain was found to exert an unusual effect, manifested as the inhibition of protein secretion by target cells. This phenomenon may be used for the express determination of cell sensitivity to colchicine-like cytostatics, including Ukrain.

[A study of the cloned human erythropoietin gene expression in mammalian cells in vitro]. / Izuchenie ékspressii klonirovannogo gena éritropoétina cheloveka v kletkakh mlekopitaiushchikh in vitro.

The human gene coding for the principal factor of erythroid cells differentiation, erythropoietin, has been isolated from the genomic phage library using an oligonucleotide probe for the gene. The construction of series of plasmids carrying the erythropoietin gene under the control of various regulatory elements is reported. Efficiency of the erythropoietin gene expression was estimated by testing of the biologically active erythropoietin in conditioned media 48 h after transient transfection of COS 1 and CHO cell lines.

[Hormonal regulation of expression of the oncogene v-src in mouse fibroblasts NIH 3T3]. / Gormonal'naia reguliatsiia ékspressii onkogena v-src v myshinykh fibroblastakh NIH 3T3.

NIH 3T3 cells were transfected by plasmid containing v-src under control of hormone-regulated LTR MMTV (pMLsrc10). This plasmid caused the foci of morphologically transformed cells. The transformed cells induced rapidly growing tumours in nude mice. In the presence of dexamethasone the efficiency of NIH 3T3 cell transformation increased ten times, while tumourigenicity remained unchanged.

[Molecular cloning of MuLV proviruses integrated into the genome of mouse erythroleukemia cells. I. Characteristics of endogenous proviruses]. / Molekuliarnoe klonirovanie provirusov MuLV, integrirovannykh v genom myshinykh éritroleikoznykh kletok. I. Kharakteristika provirusov éndogennogo proiskhozhdeniia.

The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60-70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones.

[Molecular cloning of provirus sequences of Rauscher leukemia virus from mouse erythroleukemia cell genome]. / Molekuliarnoe klonirovanie provirusnykh posledovatel'nostei leikoza Raushera iz genoma myshinykh éritroleikoznykh kletok.

Provirus from a component of Rauscher leukaemia virus (RLV) has been cloned. The provirus (the size of 5000 b. p.) contains two LTR sequences and shares expressed sequence homology with Mo-MuLV. Restriction analysis and determination of the LTR nucleotide sequence and of the site from 3'-end of proviral genome have shown the cloned provirus to be the SFEV component of RLV. LTR from this cloned provirus contains all sites necessary for transcription: CAAT and TATA sequences, "cap" site and polyadenylation signal. The LTR of the cloned provirus from SFEV component of RLV has been shown to function as a promoter in E. coli cells.

[Hormone-dependent process of the maturation of the major structural proteins of the mouse mammary tumor virus]. / Gormonozavisimyi protsess sozrevanii osnovnykh strukturnykh belkov virusa raka molochnykh zhelez myshei.

The following findings were obtained by the radio-immunoprecipitation method with antisera to gp52 and p27. When the cells were cultivated in a hormone-free medium, they contained precursors of proteins of gene gag (Pr 73gag) and gene evn (gPr 70env). No mature structural MTV proteins were found. The addition of insulin to the growth medium had no effect on maturation of protein precursors. When dexamethazone was added to the medium, gPr 70env "maturated" to gp52 of the main envelope glycoprotein of virion coat whereas Pr 73gag was not cleaved into final products. When the cells were cultivated in the presence of insulin and dexamethazone, there was a marked stimulation of Pr 73gag (although its maturation did not occur), g Pr 70env, and, especially, gp 52. Extracellular particles were found to contain p27 which was precipitated by homologous monospecific antiserum, that is, in the system of clone F2 cells processing of Pr 73gag occurred mainly in extracellular particles. Thus, expression of all three known genes (gag, pol, and env) occurred in the cells of cloned F2 culture.

Dependence of the structural MuMTV protein expression on hormone-induced murine mammary carcinoma GR/mt cell differentiation in vitro.

Effect of three hormones on the clonal cell line, originating from the stable GR/mt mammary carcinoma (MC) cell line was studied. In the hormones free media the cells had fibroblast-like shapes. Normal differentiation markers and MuMTV protein processing could not be revealed. Insulin favors epithelial cell shape and growth pattern induction, Thy 1.2 antigen expression, as it was shown by FIF test and increased clonogenicity in semisolid medium. Prolactin produces elongated, fusiform cells, milk protein synthesis and loss of clonogenicity. Both hormones exert no influence on the MuMTV protein precursors processing, as it was revealed in RIP test. Dexamethasone renders no visible influence on the cell differentiation except influence on the actin filament expression and intensification of the morphogenetic insulin action. Besides, dexamethasone switches on the env (but not the gag) product precursor processing, however, expression of the mature envelope protein on the cellular membranes occurred only in cells, entering the insulin-induced type of differentiation, as it was shown by RIP test with iodinated cells and MIF test. The gag precursor processing occurred only in extracellular virions. Delay of this process might be responsible for the preferential A-particle production by the cells described.

[Characteristics of A-type intracytoplasmic particles produced by human cells]. / Kharakteristika belkov vnutritsitoplazmaticheskikh chastits A-tipa, produtsiruemykh chelovecheskimi keltkami.

Radioimmunoprecipitation was used to analyse comparatively proteins in cytoplasmic A-type particles (CAP) and structural proteins of D-type virions in Hep-2 system of cultivated human cells. Proteins of CAP were iodinated and studied by electrophoresis in SDS-PAAG. In the preparations obtained, 60 000 (p60), 45 000, 42 000 and 20 000 dalton proteins wee detected. p60 was the main protein in CAP. It was precipitated by purified CAP antiserum rather than by antisera against structural proteins of D-type virions. It was thus demonstrated that the main structural protein of CAP Hep-2 cells contains no antigenic determinants of structural proteins of D-type oncoviruses.

[Bromodeoxyuridine-induced weakening of DNA--protein interaction in chromatin according to nucleoprotein--celite chromatography]. / Indutsirovannoe bromdezoksiuridinom oslablenie vzaimodeistvii DNK--belok v khromatine po dannym nukleoproteid-tselit-khromatografii.

After incorporation into DNA the thymidine analogue bromodeoxyuridine (BUDR) changes the cell phenotype, inhibiting specialized cellular functions. The biological activity of BUDR is suggested to be underlain by certain changes in DNA-protein interactions. It was shown by nucleo-protein-celite chromatography (NPC chromatography) of unfractionated cell lysates that interactions of BUDR-substituted DNA with proteins in Rauscher erythroleukemic cells are actually modified. In contrast to current views suggesting a strengthening of DNA-protein bonds in BUDR-treated cells, they are in fact relaxed. Rapidly labelled heterogeneous nuclear RNAs are divided by NPC chromatography into three subpopulations. The relative rates of their synthesis are significantly changed after BUDR treatment of erythroleukemic cells.