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A study was conducted of the interaction of an octadecameric oligodeoxynucleotide (dON) containing theBCL2mRNA translation start with K562 cells. Both in solution and upon lipofection, the binding of dON used at a low concentration (=30 nM) at 37°C involved two steps: saturating surface binding with the cell membrane and internalization. Three phases were revealed in the dynamics of internalization: the extent and rate of internalization increased during the first hour of incubation; decreased during the second hour; and then increased again, which was assumed to reflect the priming of new dON-binding sites. The binding constant and the number of binding sites were estimated at 10°C (the conditions preventing internalization) by consecutive dissociation of dON-cell complexes formed in 1 h at 37°C. Incubation of dON with cells led to the priming of new high-affinity binding sites and an increase of the binding constant to a level characteristic of high-affinity ligand-receptor interactions (109 M-1). High-affinity receptor-mediated binding preceded internalization of dON. Lipofection increased the binding constant and the number of binding sites severalfold but had virtually no effect on the temporal pattern and the extent of dON internalization. [ABSTRACT FROM AUTHOR]

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. Under leukemia progression differentiating Ph+, bcr/abl+ cells transform into leukemic malignant cells with differentiation block. It is assumed to be a result of subsequent mutations or activation of proliferation of long silent Ph+ cells arisen previously in the stem cells because of the translocation. Real mechanism underlying the cell transformation remains unknown. This work was performed to develop a proper cell model allowing us to study functioning of differentiating Ph+, bcr/abl+ cells and their real transformation into malignant cells with block of differentiation. For this purpose we have investigated kinetics of Ph+, bcr/abl+ cells proliferation, differentiation, cell death and transcription of antiapoptotic genes in cultured 14-day of Ph+ mononuclear cells isolated from peripheral blood of a patient in chronic phase of chronic myeloid leukemia before treatment. The results obtained revealed that Ph+ cell differentiation proceeded in accord with characteristic scheme of chronic myeloid leukemia in vivo. Myeloid cells of hematopoietic cell lineage amounted to 3/4 of live Ph+ mononuclear cells undergoing accumulation and subsequent consumption in the course of differentiation. 95% myeloid cells were differentiating Ph+ granulocytes. The most deal of differentiating Ph+ cells was myelocytes. The rate ratio of myelocyte accumulation to its subsequent consumption showed that the rate of transformation into metamyelocytes was significantly decreased at this differentiation stage. Ph+ cells cultivation curves characterized cell death at different differentiation stages. There were observed the cell death of proliferating Ph+ cells and Ph+ myelocytes, and intensive death of mature cells as well. P/D index, that is ratio of immature Ph+ granulocytes differentiated by cell dividing (blasts, promyelocytes and myelocytes) to the cells differentiated without dividing (metamyelocytes and mature neutrofiles), revealed active of proliferation at the beginning of cultivation and unexpected new proliferative activity at the end of cultivation in the presence of growth factor. The peaks of antiapoptotic bcr/abl gene transcription activity coincided with the observed active proliferation at the beginning and at the end of cultivation. Cell proliferation, differentiation and apoptosis were noticeably accelerated by growth factor treatment. Thus, the study of the Ph+ cells cultivation kinetics is rather informative approach to investigation of continuous regulation of cellular and molecular processes in vitro in the case of chronic myeloid leukemia and allows more complete consideration of Ph+, bcr/abl+ cells hematopoiesis.

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. The enhanced extent and rate of oligonucleotide internalization was diminished after first hour incubation and later they were increased again. This reflected rising additional binding sites that provided internalization. At chosen time-points the internalization of ODN into cells, been proceeded at 37 degrees C, were at most abruptly abrogated by cooling down. ODN to K562 cell membrane binding constants and specific number of binding sites have been determined. Time-intervals, providing equilibrium for each successive stage of multistep ODN bound/free determination, were maintained. It was established that receptor binding with increased binding constant (more than 2 x 10(9) M(-1)) promoted ODN internalization. Oligonucleotide binding and internalization with prolonged incubation were also up-regulated due to priming new binding sites of higher affinity. Lipofection enhanced ODN binding to cell membrane but conserved the main features of ligand-receptor interaction. During lipofection constants and ODN binding site numbers increased without changing the overall time-pattern of the process, observed for ODN without liposomes. Extent and rate of internalization of ODN in liposomal formulation did not differ substantially from ODN in solution.

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. In some of the cell lines, an inhibition of transcription of the E1 oncogenes was observed. The normalization also produced cells that divided 2 - 5 times and died and cells that reverted to a transformed phenotype in 2 - 10 days. The latter appeared predominantly upon the action of the antisense oligonucleotides.

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. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.

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. The data obtained assumes the mechanism of the derivatives transport to be liquid endocytosis. Being distributed in the cell components the polyalkylating derivatives were accumulated by the cell nuclei up to 10(5)-10(7) molecules per nucleus. Transport efficiency is much greater in the anchored cells than in the suspended ones. Though essential dephosphorylation of the utilized substances is observed in the SH2 cells, part of them maintain native chain length and the 5'-phosphate group after 1 h incubation in nucleic acids obtained from the cell nuclei.

It was shown that the buoyant density of DNA isolated from lymphocyte incubation media as well as from blood plasma of healthy patients and patients with chronic lympholeukemia (CLL) using the SDS-phenol method with subsequent ultracentrifugation in a cesium trifluoroacetate density gradient is 1.59-1.60 and 1.60-1.63 g/ml, respectively. Using electrophoresis in 0.6% agarose gel, it was found that the DNA excreted by lymphocytes from healthy patients and CLL patients is a high molecular weight fraction comprising 21,000 nucleotide pairs. This DNA fragment contains 90-95% of [3H]thymidine used for the labeling of DNA excreted by lymphocytes from healthy patients and patients with CLL. No traces of [3H]thymidine were found in the middle part of the agarose gel containing the diffuse material bound to ethidium bromide.

The action of calmodulin (CM) given in doses of 10(-5)-10(-8) M on lymphocyte proliferation in healthy subjects was studied. All the test doses of CM produced a stimulant effect 72 h after the lymphocyte incubation was commenced. The rate of 3H-thymidine incorporation experimentally was 1.5-3 times higher than in the control. It is assumed that the release of CM in the foci of tissue injury or inflammation may serve one of the factors stimulating lymphocyte proliferation in an individual focus.

Possible use of ethidium bromide immobilized on polyacrylamide gel for DNA extraction from biological media, e.g., cell lysates and blood serum, is discussed. The ability of the affinity gel to cause fractionation of single- and double-stranded DNA is demonstrated.

Fluorometric measurements of the levels of radiation injury to DNA of leucocyte lysates were made on the basis of the estimates of alkaline denaturation rate obtained by the method of Birnboim and Jevcak. Leucocytes of human blood in vitro and rats were exposed to doses of 0.5, 1, 1.5, 3, 6 and 9 Gy. It was shown that the percentage of double-stranded DNA of leucocytes of irradiated human blood decreased with the increase in the radiation dose while that of exposed rats decreased immediately after irradiation and was normalized after 20 h.