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

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.

Gene Expression upon Proliferation and Differentiation of Hematopoietic Cells with PH Chromosome ex vivo

“…The kinetic curves of gene expression were compared to the regularities of the proliferation and differentiation of the granulocyte populations, the apoptosis and distribution of Ph + cells in the phases of the cell cycle, with alternating the proliferation and maturation stages, which regulate the P/D index efficiency of these processes [1– 4 ] and are given in a polynomial approximation. The regularities of the proliferation and differentiation of Ph + cells have already been studied [1– 6 ]; the polynomial approximation of these curves is considered here, since they fail to obey either the logarithmic or exponential law, and the kinetic curves corresponding to these dependences have several maxima and minima ( Figs.…”

“…The characteristics of the Ph + cells and CML patients from whose PB and BM the mononuclears were isolated are given in [2– 5 ]. The types of bcr/abl mRNA (b3a2, b2a2 or e1a2) in the Ph + cells were determined by RT-PCR [ 2 , 5 ].…”

“…The degree of apoptosis and distribution of cultured Ph + cells over the phases of the cell cycle were analyzed cytofluorometrically [1– 4 ] in the granulocyte gate on an EPICS-XL flow fluorimeter. Ph + cell samples (5,000 cells each) isolated from BM and PB in a Ficoll density gradient and the samples collected during the cultivation were centrifuged for 7 min at 600 g and 4 о С, washed with PBS, and fixed dropwise adding cooled 70% ethanol during 30 min at 4 о С.…”

“…The kinetic curves of the P/D efficiency index (the ratio between the neutrophil proliferation and maturation rates) were obtained as ratios between the accumulation of immature proliferating cells, P (blasts, promyelocytes, myelocytes), and the accumulation of neutrophils maturing without dividing, mature cells, D (metamyelocytes, bands and segments) according to [1– 4 ].…”

“…The proliferation rate is higher than the maturation rate at stage 1, whereas the maturation rate is higher at stage 2. The alternation of the stages and their rates maintains the optimal level of proliferation and differentiation efficiency in Ph + cells [1– 4 ] and determines the wave-like regulation of these processes.…”