Research Article: Cell Regulation of Proliferation and Differentiation ex vivo for Cells Containing Ph Chromosome in Chronic Myeloid Leukemia

Date Published: October , 2009

Publisher: A.I. Gordeyev

Author(s): N.I. Grineva, T.V. Akhlynina, L.P. Gerasimova, T.E. Manakova, N.G. Sarycheva, D.A. Schmarov, A.M. Tumofeev, N.M. Nydenova, L.Yu Kolosova, T.I. Kolosheynova, L.G. Kovaleva, S.V. Kuznetsov, A.V. Vorontsova, A.G. Turkina.

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Abstract

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/D1 = 1-20 or P/D2 ⇐ 1. At the same time period, the proliferation and differentiation of the Ph+ cells type 3 proceeds with repeated alternations of stages with P/D1 = 1-4 or P/D2 ⇐ 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.

Partial Text

Leukemias accounts for 1% of all deaths and 4-10% of deaths from cancer. The prevalence of leukemias and lymphomas varies from 3 to 9:100 000, depending on the geographical region. Unfavorable radiation and ecological environment can increase it by 1.5 logs. In the U.S., leukemias are the major reason of death in children before 15.

Materials: heparin (Flow, UK); Limphoprep, alpha-MEM media (MP Biomedical, USA); DEPC, Hepes, Tris, PBS, fetal bovine serum (FBS), sodium citrate, laurilsarcosyl (ICN, USA); trypan blue stain, L-glutamine and 2-mercaptoethanol (Serva, Germany), penicillin and streptomycin (OAO “Biochimik” Saransk Russia); G-CSF (F.Hoffmann-La Roche Ltd, France); tabletted PBS ( mM”>10 mM phosphate buffer+ 0,13 M NaCl + 2,7 mM KCl, pH 7,4), NPZ “EKO-servise” Russia.

In order to study the differences in PAmp;D in the Ph+ cell cultures of patients with different CML phases, we obtained kinetic plots for 34 samples of Ph+ mononuclear cells from the PB and BM of 23 CML patients under cultivation in strictly identical conditions. Ph+ mononuclear cells from PB and BM contain hematopoietic cells capable of self-renewal at PAmp;D and to PAmp;D for 2-3 cycles, forming the full set of Ph+ cells [33, 39].

Myelopoiesis (PAmp;D of myeloid division of hematopoiesis) begins with the proliferation and differentiation of hematopoietic progenitor cells and their immature progenies. Then PAmp;D continues with neutrophils maturation without dividing [3, 40, 41]. In this study, we obtained and analyzed ex vivo PAmp;D kinetic plots in 34 samples of Ph+ cells from the PB and BM of 23 CML patients. They demonstrated evident differences in the PAmp;D of Ph+ cells from individual CML patients.

The elicited PAmp;D regulation in Ph+ cells ex vivo reveals the cellular aspects of genetic PAmp;D regulation. As was shown in the present work, cellular regulation is mediated by alternation of Ph+ cells proliferation with efficient maturation and is accomplished by proliferation inhibition by maturating without dividing neutrophils – progenies of the same proliferating Ph+ progenitor cells under conditions of apoptosis blockage.

1. The PAmp;D regulation of Ph+ cells from different CML patients is performed by alternation of effective proliferation with effective maturation. In the maturation stage, the Ph+ cells proliferation is inhibited by accumulating neutrophils with apoptosis blockage interrupting proliferation and maintaining the optimum PAmp;D level.

This work was supported by the Russian Foundation for Basic Research (grant no. 06 04-08372-ofi).

 

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