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.

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph + ) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr / abl , bax , and bcl2 . The regulation of apoptosis in main types of Ph + cells depends on the alternation of (1) Ph + cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr / abl expression and expression of bcl2 > bax . A stable block of apoptosis is associated with CML progression and in Ph + cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph + cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.

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. Expression of these genes decreases to a considerable level under alternation of the Ph(+)cell proliferation and maturation stages and whenever the expression is greatly diminished under significant neutrophil accumulation and especially under repeated alternation of the stages. In the course of neutrophil maturation, gene expression levels decrease in the range of gapdh > actin > c-myc, bcr/abl,p21 > p53 > bcl2 > bax.The expression levels of these genes in neutrophils are lower than those in myelocytes and lower by an order of magnitude than that in the cells with a prolonged proliferation stage. TheBcr/ablexpression gene under prolonged maturation and neutrophil accumulation is inhibited; however it is enhanced by 2-3 times for the proliferation stage with myelocyte accumulation. Minimalbcr/ablexpression is observed under overexpression ofp53, mdm2, p21, c-myc,as well as under cell maximum at the S and G2/M phases. Bcr/abloverexpression is observed under low expression of thep53, p21, mdm2genes. In the Ph(+ )cells with a high P/D efficiency index (5-20), overexpression of the genes in the range ofbcr> gapdh>bcr/abl, as well as a decreased expression of thep53, bcl2, mdm2, p21<< gapdh genes is observed for Ph(+)cells from the CML blast crisis and CML acceleration phase. Low control of cell proliferation and cell cycle by gene-regulators presumably promotesbcr/abloverexpression and activаtes the production ofbcr/abl+ cells. Apoptosis in the Ph(+ )cells is induced by expression of thebax > bcl2, р53, p21, c-myc andgapdhgenes. The blocking of Ph(+)cell apoptosis, neutrophil accumulation, and decrease in the expression of the p53, mdm2 and p21, c-myc,bcr/abl genes occur at the maturation stage.

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. The kinetic plots of alternations pass through control points of crossing plots, where the parameters of proliferation and maturation are equal. The indices of P/D efficiency (ratio of proliferation and maturation rates) are 1.06±0.23 and don't depend on time, alternation order, or sources of Ph(+) cells - CML patients. During stages alternation, conversely, the parameters of Ph+ cell proliferation and maturation vary. The proliferation stages are characterized by increased proliferating cells content, a decreased number of neutrophils, and apoptosis induction. At the maturation stages, conversely, apoptosis is inhibited, the number of mature neutrophils increases, while immature Ph(+) cells decrease. High content neutrophils inhibit the proliferation of Ph(+) cells and impair their own maturation by inversion of maturation order, probably through a feedback mechanism. The regulation differences ex vivo reveal three types of Ph(+) cells from various individual CML patients, distinguished by the number and duration of alternating stages of proliferation and maturation. Ph(+) cells types 1 and 2 have one prolonged stage of effective proliferation or effective maturation with efficiency indices P/D(1) = 1-20 or P/D(2) ⇐ 1. At the same time period, the proliferation and differentiation of the Ph(+) cells type 3 proceeds with repeated alternations of stages with P/D(1) = 1-4 or P/D(2) ⇐ 1. Type 1 Ph(+) cells (~20%) were isolated from patients in advanced stages of CML, while Ph(+) cells types 2 and 3 (30 and 50% correspondingly) were isolated from CML chronic phase patients sensitive to chemotherapy.

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.

A study was conducted of the interaction of an octadecameric oligodeoxynucleotide (dON) containing the BCL2 mRNA 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 (10 9 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.

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.

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. At low concentration ODN bound efficiently with K562 and penetrated into the cells via binding cell surface with rather high affinity and priming new binding sites. The loose binding constant at 4 degrees C was 1.8 x 10(9) M(-1) both for binding ODN in solution and ODN-associated liposome. The number of loose binding sites under both treatments was rather high: 4.6 to 6.6 pmoles per 10(6) cells. The extent of ODN penetration into the cells showed higher potential site numbers than initially seen and reached 8.6 pmoles per 10(6) cells for four hours incubation at 37 degrees C.

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.

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 1.1 clone was strongly altered. Subcloning of a fragment of the altered region and blot hybridization analysis using it as a DNA probe revealed recombination in the 3' region of the Mbcr locus in clone lambda BCR 1.1 which resulted in insertion of unknown sequences into the region. A modified system is suggested for chromosome 22 breakpoint identification using restriction analysis of genome DNA with four restriction endonucleases and one 5'-DNA probe.

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.