Abstract
Previous work has demonstrated that phorbol ester (TPA)-induced adherence of human U937 myeloid leukemia cells can be blocked upon down-modulation of the β2-integrin CD11b after stable transfection of U937 cells with a pMTH1 vector-containing the CD11b gene in antisense orientation (asCD11b-U937) [Otte et al., (2011)]. In the present study, alterations in metabolism-associated factors, particularly intra- and extracellular proteases were investigated. A measurement of telomerase activity in the leukemic cells revealed continuously decreasing telomere adducts within 72 h of TPA treatment in pMTH1-U937 cells. In contrast, telomerase activity sustained in asCD11b-U937 upon TPA-induced differentiation. Flow cytometric analysis confirmed unchanged CD11b levels in TPA-induced asCD11b-U937 in contrast to elevated levels in pMTH1-U937 whereby the expression of other β2-integrins including CD11a, CD11c and CD18 was increased in both populations after TPA treatment. Moreover, adherent pMTH1-U937 demonstrated the expression of monocytic differentiation markers including F4-80 and CD14 and an increased MIP-1α production which remained at low or undetectable in TPA-induced asCD11b-U937. These effects indicated an altered response of the different cell populations to the TPA-induced differentiation process. Indeed, Western blot analysis revealed differences in the expression levels of intracellular metabolic factors including MnSOD and p97/VCP and after measurement of 20 S proteasomal proteolytic activity. In addition, increased levels of extracellular metabolic factors including the matrix metalloproteinases MMP-1, MMP-7 and MMP-9 were observed in pMTH1-U937 cells in contrast to unaltered levels in asCD11b-U937 cells.
Highlights
The human U937 myeloid leukemia cell line represents an in vitro model for monocyte/macrophage-like differentiation and retrodifferentiation [1,2,3,4,5]
The proliferation of wild-type U937 and pMTH1U937 cells as measured by [3 H]thymidine incorporation revealed a significant reduction by about 75% to 85% after 72 h of TPA treatment (Figure 1b) which is in agreement with previous studies demonstrating TPAmediated growth inhibition [1,10,11]
Phorbol ester treatment of non-adherent human myeloid leukemia cells is associated with cell attachment to form 3-dimensional cell aggregates, growth arrest and monocytic differentiation, and the reversible process of retrodifferentiation and rejuvenation reveals the loss of previously acquired monocytic features, a regained proliferative capacity and cell detachment to form again a single cell suspension of monoblastoid precursor cells [4,5]
Summary
The human U937 myeloid leukemia cell line represents an in vitro model for monocyte/macrophage-like differentiation and retrodifferentiation [1,2,3,4,5]. Previous work has demonstrated that a differentiationdefective subclone of the U937 cell line, termed TUR (TPA-U937-resistant), fails to express significant levels of CD11b after TPA treatment [10] These human TUR leukemia cells are unable to attach and continue to proliferate in response to a phorbol ester stimulation [11] indicating that CD11b displays a differentiationassociated function beyond an involvement in the regulation of cell attachment. Previous work has demonstrated that a down-modulation of the CD11b integrin fails to develop certain markers of a monocytic phenotype following exposure to the differentiationinducing TPA [1,12] Such a differentiation program along the monocyte/macrophage-like lineage in TPA-treated U937 cells requires significant metabolic changes and other studies have shown that this process is accompanied by alterations in the expression and activity of metabolizing factors including the 20 S proteasome [13], manganese peroxidase [14] and the valosin-containing protein VCP/p97 [7]. The acquired adherence of myelocytic cells during monocytic maturation is paralleld by a restructure of the extracellular matrix involving a variety of matrix metalloproteinases such as MMP-1, MMP-7 and MMP-9 [14]
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