Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

Abstract

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.

Animal models of acute myelogenous leukaemia – development, application and future perspectives

“…Prominent examples include differentiation induction therapy, developed from preliminary studies with cyclic adenosine monophosphate (cAMP) inducing cholera toxin in the BNML and BNML-cholera toxin-resistant models, 161,268 269 circuitously leading to the clinical studies of agents such as theophylline 273 (cAMP induction/stabilisation), Gemtuzumab ozogamicin 274 (conjugated mAb) and granulocyte colony-stimulating factor 275 (G-CSF-cytokine). These results and the application of the BNML rat model in more recent work pertaining to angiogenesis and microenvironmental modifications by Iversen et al [276][277][278][279] rationalise continued interest in this model.…”

Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1α

“…The lack of oxygen in BM is possibly further aggravated with anemia that often accompanies newly diagnosed AML patients. In fact, Jensen et al 15 have reported that hypoxic fraction of leukemic cells and normal cells inoculated in the Brown Norwegian rat increase significantly in the BM. On the other hand, leukemic cells are cultured in vitro at ambient oxygen (21%) in most circumstances, while in vivo cells are physiologically exposed under much lower oxygen levels ranging from 16% in pulmonary alveoli to less than 6% in most peripheral organs of the body.…”

Severe Hypoxia Defines Heterogeneity and Selects Highly Immature Progenitors Within Clonal Erythroleukemia Cells

“…Although these concepts have been developed for solid tumors, oxygen levels also may be particularly low around leukemia cells. Indeed, bone marrow, which is physiologically characterized by a high level of cell crowding and poor vascularization [1], can undergo a further reduction of oxygenation in patients with leukemia [10] as a result of the fast growth of leukemia cells and anemia, which commonly develops as a consequence of leukemic growth.…”