[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. The way is not described early. Neutrophils accumulation correlates with apoprosis blocking. Apoptosis level enhances under neutrophils exhausted. Apoptosis blockage allows cells to proliferate and, thus, to form new portion of neutrophils with consecutive regular their death as well a consequent alternation of apoptosis blocking and inducing. This way regulates proliferation efficiency indexes P/D that reflect Ph+ cells proliferating potential and performs cycle completion for proliferation and differentiation. The second way of apoptosis regulation starts from proliferation stage and performs for 1/2/1 alternations under diminished content of neutrophils and a little increase under next maturation. It leads to resistant depressed apoptosis levels that, at maximal points, are 3-8 times lower than those under alternation 2/1/2. Resistant apoptosis blocking is observed in the Ph+ cells with prolong proliferation or maturation stages, when blasts and myelosytes are accumulated under enhanced bcr/abl and bcl2 > box gene expression and remain under next maturation. Stable apoptosis blocking is accompanied by increasing amounts of blasts and myelocytes and enhancing bcr/abl and bcl 2 > bax expression. This is observed under CML progression. Ph+ cells cultivation may be useful for more distinct diagnostics of CML phases of individual CML patients and optimization of the treatment.

[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. 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.

[Circular dichroism study of the reaction between the protein product of bacteriophage f1 gene 5 and synthetic polynucleotides]. / Izuchenie vzaimodeistviia belka--produkta gena 5 bakteriofaga f1 s sinteticheskimi polinukleotidami metodom krugovogo dikhroizma.

The complexes of gene 5 protein (phage f1) with single-stranded and double-stranded polynucleotides were investigated by circular dichroism (CD). In our experiments the concentration of the protein varied accordingly to polynucleotide. A decrease of the CD amplitude for polynucleotide-protein complexes was shown in all the regions of wavelength studied. The data indicate that the protein is bounded to the polynucleotide. This protein bounds differently to double-stranded polynucleotides containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains. This difference is explained by differences in the form of the secondary structure of polynucleotides, containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains.

[Effect of the protein product of phage f1, gene 5, on heat denaturation of synthetic polynucleotides]. / Vliianie belka--produkta gena 5 faga f1--na teplovuiu denaturatsiiu sinteticheskikh polinukleotidov.

The influence of phage f1 gene 5 protein on melting of the synthetic polynucleotides has been investigated, using UV-spectroscopy. In our experiments we have varied the proteins concentration. It has been shown, that the protein lowers the melting temperature of the studied polynucleotides (d/A--Tn dAndTn, rAndTn, rAn.r n, dAn.rn). The melting temperatures and the shapes of melting curves of various polynucleotides differ when the same protein concentrations are used. We have shown that the protein binds to the double-stranded polynucleotides, containing ribo-ribo-, deoxyribo-ribo-chains. The difference in melting temperatures and shapes of melting curves was explained using the data about the differences in the secondary structure of these polynucleotides. Only for d/A-Tn renaturation was observed after sample cooling. It may reflect the single-stranded hairpin structure of this polynucleotide.

[Influence of protein bound with single-stranded DNA on the synthesis of RNA and poly(A). III. Protein product of F1 gene 5]. / Vliianie na sintez RNK i poli (A) belkov, sviazyvaiushchikhsia s odnonitchatoi DNK. III. Belok--produkt gena 5 faga fl.

Influence of the protein product of F1 phage gene 5 (protein 5) on the synthesis RNA and poly(A) in vitro was studied. It has been shown, that protein 5 has no effect on the transcription of the native DNA by E. coli RNA-polymerase, but completely prevents RNA and poly(A) synthesis on the denatured or single-stranded DNA at the protein/DNA ratio 10:1. Protein 5 inhibits poly(A) synthesis with oligo(dT)9 and oligo(dT)12 as a template, preventing binding of enzyme to the oligonucleotide. After the initiation of the poly(A) synthesis the inhibition becomes considerably wearer. The biological function of the inhibition of the transcription by "unwinding" proteins is discussed.