Abstract
BackgroundRetrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.ResultsSeparation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G0/G1 cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G0/G1 growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.ConclusionThe findings demonstrated that monocytic differentiation and G0/G1 growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.
Highlights
Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors
After 2–3 weeks in the absence of TPA, the differentiated population down-modulates all markers acquired during the previous differentiation process and re-enters the proliferative cell cycle by a retrodifferentiation program, which results in the undifferentiated phenotype [6,7,8]
In order to investigate the expression of valosin-containing protein (VCP)/p97 during the cell cycle, centrifugal elutriations of proliferating U937 cells were performed to separate cells within distinct phases of the cell cycle (Fig. 1)
Summary
Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The human myeloid leukemia cell line U937 represents a well-characterized in vitro tumor model of differentiation and retrodifferentiation, which can be induced to differentiate along the monocyte/macrophage-like pathway after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) [1]. During this differentiation process, the autonomously proliferating tumor cells become adherent, undergo a complete growth arrest and acquire a variety of morphological and functional changes typical for normal monocytes and macrophages [1,2,3,4,5]. Since fundamental cellular functions are altered and reverted in this differentiation and retrodifferentiation model [11], the precise signaling pathways for the coordination of this phenomenon remain unclear and may involve further regulatory compounds of the proteasome, including certain ATPases
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