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The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97.

Bertram C, von Neuhoff N, Skawran B, Steinemann D, Schlegelberger B, Hass R - BMC Cell Biol. (2008)

Bottom Line: 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.Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Gynecology, Biochemistry and Tumor Biology Lab, Medical School Hannover, Carl-Neuberg-Str, 1, D - 30625 Hannover, Germany. bertram.catharina@mh-hannover.de

ABSTRACT

Background: Retrodifferentiation 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.

Results: Separation 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.

Conclusion: The 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.

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Description of off-target effects during RNAi of VCP/p97. Off-target effects reflect VCP/p97-siRNA-independent modulations of distinct genes. Only genes that were affected by both chosen VCP/p97-siRNAs were considered as down- or up-regulated.
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Figure 7: Description of off-target effects during RNAi of VCP/p97. Off-target effects reflect VCP/p97-siRNA-independent modulations of distinct genes. Only genes that were affected by both chosen VCP/p97-siRNAs were considered as down- or up-regulated.

Mentions: A possible functional role of VCP/p97 in differentiating U937 cells was addressed by a siRNA approach. The transfection method was tested with a FITC-conjugated control siRNA which revealed a transfection efficiency in U937 cells of >95% as evaluated by FACS analysis (Fig. 6A). The successful down-modulation of VCP/p97 protein in VCP-siRNA-transfected cells was demonstrated in appropriate Western blots for both, U937 control and TPA-differentiating cells (Fig. 6B). Functional changes in VCP-siRNA-transfected cells following phorbol ester-induced monocytic differentiation have been examind by DNA microarray analysis (Fig. 6C, D). Thus, differentiation-associated genes, which are elevated during myelomonocytic maturation, including the cytokine receptor for interleukin-1, were expressed at significantly reduced levels in TPA-treated VCP/p97-siRNA-transfected U937 cells. According to the cell attachment as a consequence of differentiation along the monocytic/macrophage-like pathway, genes associated with the formation of the cytoskeleton (e.g. adducin1) and certain extracellular matrix related genes such as ADAM metalloproteinase 13 and the extracellular glycoprotein fibronectin showed a decreased expression due to VCP/p97 RNAi (Fig. 6C). Moreover, previous work has demonstrated that TPA-mediated signals in U937 cells are relayed via activation of protein kinase C and downstream kinases to activate appropriate transcription factors for the induction of differentiation-associated genes [8]. In this context, pleckstrin homology domain containing proteins, a MAP kinase interacting protein and phospholipase C were significantly down-modulated in TPA-treated VCP-siRNA-transfectants (Fig. 6C). With respect to genes involved in protein metabolism and particularly in transport functions, sulfotransferase 2A, an ATPase (ATP8B3), a vesicle transport protein (SFT2D3) and a Golgi-associated gene (COG1) were reduced expressed (Fig. 6C). In contrast, the KDEL receptor was up-regulated, which is responsible for the retention of newly synthesized proteins during the quality control in the ER [32] (Fig 6D). Moreover, metabolic compounds involved in post-translational modifications in the lysosome and microsome such as β-glucoronidase, arylacetamide deacetylase, phosphoserine aminotransferase and xylosysltransferase were increased expressed (Fig. 6D). TPA-induced differentiation is also associated with growth arrest and down-modulation of cell cycle-regulatory factors [9,10]. In contrast, TPA treatment of VCP-siRNA-transfected U937 cells revealed an increased expression of the transcriptional regulator ETV6 and certain histones. (Fig. 6D). However, siRNA-targeting in cells also involves off-target effects, which reflect VCP/p97-siRNA-independent modulations and some of these genes are listed in Figure 7.


The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97.

Bertram C, von Neuhoff N, Skawran B, Steinemann D, Schlegelberger B, Hass R - BMC Cell Biol. (2008)

Description of off-target effects during RNAi of VCP/p97. Off-target effects reflect VCP/p97-siRNA-independent modulations of distinct genes. Only genes that were affected by both chosen VCP/p97-siRNAs were considered as down- or up-regulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277395&req=5

Figure 7: Description of off-target effects during RNAi of VCP/p97. Off-target effects reflect VCP/p97-siRNA-independent modulations of distinct genes. Only genes that were affected by both chosen VCP/p97-siRNAs were considered as down- or up-regulated.
Mentions: A possible functional role of VCP/p97 in differentiating U937 cells was addressed by a siRNA approach. The transfection method was tested with a FITC-conjugated control siRNA which revealed a transfection efficiency in U937 cells of >95% as evaluated by FACS analysis (Fig. 6A). The successful down-modulation of VCP/p97 protein in VCP-siRNA-transfected cells was demonstrated in appropriate Western blots for both, U937 control and TPA-differentiating cells (Fig. 6B). Functional changes in VCP-siRNA-transfected cells following phorbol ester-induced monocytic differentiation have been examind by DNA microarray analysis (Fig. 6C, D). Thus, differentiation-associated genes, which are elevated during myelomonocytic maturation, including the cytokine receptor for interleukin-1, were expressed at significantly reduced levels in TPA-treated VCP/p97-siRNA-transfected U937 cells. According to the cell attachment as a consequence of differentiation along the monocytic/macrophage-like pathway, genes associated with the formation of the cytoskeleton (e.g. adducin1) and certain extracellular matrix related genes such as ADAM metalloproteinase 13 and the extracellular glycoprotein fibronectin showed a decreased expression due to VCP/p97 RNAi (Fig. 6C). Moreover, previous work has demonstrated that TPA-mediated signals in U937 cells are relayed via activation of protein kinase C and downstream kinases to activate appropriate transcription factors for the induction of differentiation-associated genes [8]. In this context, pleckstrin homology domain containing proteins, a MAP kinase interacting protein and phospholipase C were significantly down-modulated in TPA-treated VCP-siRNA-transfectants (Fig. 6C). With respect to genes involved in protein metabolism and particularly in transport functions, sulfotransferase 2A, an ATPase (ATP8B3), a vesicle transport protein (SFT2D3) and a Golgi-associated gene (COG1) were reduced expressed (Fig. 6C). In contrast, the KDEL receptor was up-regulated, which is responsible for the retention of newly synthesized proteins during the quality control in the ER [32] (Fig 6D). Moreover, metabolic compounds involved in post-translational modifications in the lysosome and microsome such as β-glucoronidase, arylacetamide deacetylase, phosphoserine aminotransferase and xylosysltransferase were increased expressed (Fig. 6D). TPA-induced differentiation is also associated with growth arrest and down-modulation of cell cycle-regulatory factors [9,10]. In contrast, TPA treatment of VCP-siRNA-transfected U937 cells revealed an increased expression of the transcriptional regulator ETV6 and certain histones. (Fig. 6D). However, siRNA-targeting in cells also involves off-target effects, which reflect VCP/p97-siRNA-independent modulations and some of these genes are listed in Figure 7.

Bottom Line: 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.Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Gynecology, Biochemistry and Tumor Biology Lab, Medical School Hannover, Carl-Neuberg-Str, 1, D - 30625 Hannover, Germany. bertram.catharina@mh-hannover.de

ABSTRACT

Background: Retrodifferentiation 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.

Results: Separation 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.

Conclusion: The 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.

Show MeSH
Related in: MedlinePlus