KINETICS OF PROLIFERATION, DIFFERENTIATION AND TRANSCRIPTION OF GENES REGULATING APOPTOSIS IN CULTURED HUMAN
BCR/ABL+Ph+ CELLS
O. V. Akhlynina, L. P. Gerasimova, G. P. Sarkisyan, T. V. Borovkova, E. A. Dukhovenskaya,
T. E. Manakova, N. M. Naydenova, A. M. Timofeev, N. I. Grineva 1
Research Center for Hematology RAMS, Moscow;
1 e-mail: ngrineva@blood.ru
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 Pha 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 Pha 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 Pha myelocytes,
and intensive death of mature cells as well. P/D index, that is ratio of immature Pha 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.
Key words: hematopoietic Ph+ cells in culture, kinetics of cell proliferation,
differentiation and apoptosis in vitro, RT-PCR bcr/abl, bcl2, mdm2, p53 mRNA in the total RNA
of the Ph+ cells under chronic myeloid leukemia
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