Original Manuscript

Leukemia (2005) 19, 1439–1445. doi:10.1038/sj.leu.2403826; published online 2 June 2005

Differentiation

SET-induced calcium signaling and MAPK/ERK pathway activation mediate dendritic cell-like differentiation of U937 cells

A Kandilci1 and G C Grosveld1

1Department of Genetics and Tumor Cell Biology, Mail Stop 331, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA

Correspondence: Dr GC Grosveld, Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. Fax: +1 901 526 2907; E-mail: gerard.grosveld@stjude.org

Received 18 February 2005; Accepted 29 April 2005; Published online 2 June 2005.

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Abstract

Human SET, a target of chromosomal translocation in human leukemia encodes a highly conserved, ubiquitously expressed, nuclear phosphoprotein. SET mediates many functions including chromatin remodeling, transcription, apoptosis and cell cycle control. We report that overexpression of SET directs differentiation of the human promonocytic cell line U937 along the dendritic cell (DC) pathway, as cells display typical morphologic changes associated with DC fate and express the DC surface markers CD11b and CD86. Differentiation occurs via a calcium-dependent mechanism involving the CaMKII and MAPK/ERK pathways. Similar responses are elicited by interferon-italic gamma (IFN-italic gamma) treatment with the distinction that IFN-italic gamma signaling activates the DNA-binding activity of STAT1 whereas SET overexpression does not. In addition, unlike IFN-italic gamma signaling, SET generated stress-induced p38/MAPK activity. Interestingly, IFN-italic gamma treatment transiently upregulated endogenous SET in a dose-dependent manner. These results suggest that SET is part of both IFN-italic gamma-mediated and stress-mediated cellular responses and that SET induces cell differentiation via calcium and MAPK/ERK pathways.

Keywords:

SET, dendritic cell differentiation, Ca2+ signaling, IFN-italic gamma signaling

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Introduction

The human SET gene, a member of the NAP/SET family,1 is located on chromosome 9q34. SET was initially identified as one partner in the SET-CAN fusion gene, which results from the t(9;9)(q34;q34).2 SET encodes a 39-kDa ubiquitously expressed, predominantly nuclear phosphoprotein. One-third of its C-terminal acidic amino acids form an acidic tail.2, 3, 4 SET (also known as template-activating factor I beta (TAF-Ibeta)) physically interacts with several protein complexes, which suggests that it has diverse functions including Granzyme A–induced apoptosis,5, 6 chromosome remodeling,7 transcriptional regulation,8 mRNA stabilization9 and cell cycle regulation.10, 11, 12, 13

SET also forms a complex with the MLL leukemic fusion protein and type-2A protein phosphatase (PP2A).14 Among other functions, PP2A regulates cell cycle progression,15, 16 and one target of PP2A is the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) pathway.17 SET inhibits PP2A activity,10, 12 and overexpression of SET activates MAPK and prevents Fas-mediated apoptosis.17

It has been proposed that SET has opposite functions during cell cycle progression:13, 18 First, SET binds p21. This interaction reverses the inhibitory effect of p21 on cyclin E–CDK2 complexes and suggests a positive regulatory role for SET on G1/S transition by allowing cyclin E–CDK2 activity in the presence of p21.11 Second, SET interacts with cyclin B–CDK1.19 Overexpression of SET inhibits cyclin B-CDK1 activity, which in turn, blocks the G2/M transition; this finding suggests a negative regulatory role for SET in G2/M transition.13 Overexpression of cell division autoantigen-1 (CDA1), another member of the NAP/SET family, inhibits proliferation and decreases bromodeoxyuridine uptake in HeLa cells.20 Acidic and basic domains of CDA1 show 40% identity and 68% similarity to SET.20

We have recently shown that overexpression of SET in the human promonocytic cell line U937 causes G0/G1 arrest and stimulates differentiation, an effect dependent on the acidic domain of SET.21 Therefore, the level of expression of SET may affect cellular processes, as determined by cell type and context. Herein, we further analyzed the mechanisms of SET-induced differentiation of U937 cells and showed that it occurs along a dendritic pathway as a result of calcium signaling and MAPK/ERK activation.

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Materials and methods

Cell culture and FACS analysis

Stable U937 cell clones expressing tetracycline-regulatable Flag-epitope tagged SET (FS) were generated and maintained as described previously.21 Cells that were either treated or non-treated with vitamin D3 (10-7 M) were cultured in the presence (FS-uninduced) or absence of tetracycline (FS-induced) after initial washing (tet[+] cells with tet[+] medium and tet[-] cells with tet[-] medium) and the expression of CD11b (Beckman Coulter Inc., Fullerton, CA, USA), CD14, CD86, CD83, CD80 and HLA-DR (all from BD Biosciences, Franklin Lakes, NJ, USA) were analyzed by FACS at day 4 of culture. For inhibition assays, cells were treated with specific inhibitors (CsA (2 mug/ml; Sigma, St Louis, MO, USA), W-7 (15 muM; Calbiochem, San Diego, CA, USA), KN-93 (3 muM; Calbiochem) SB202190 (5 muM; Upstate, Charlottesville, VA, USA), PD98059 (50 muM; Cell Signaling Technology, Beverly, MA, USA) or DMSO vehicle as a control. At day 4 of culture, each sample was divided into three aliquots, which were used for light microscopy, FACS and immunohistochemical analysis. For light microscopy, 1x106 cells were resuspended in 2 ml serum-free RPMI, transferred onto polylysine-coated six well plates (Becton Dickinson Labware, MA, USA) and cultured for 1 additional hour. Cells were photographed with an Olympus IX-70 inverted microscope. All the inhibitors were first tested for toxicity and optimized nontoxic doses were used in the further experiments.

Microarray and data analysis

Total RNA was isolated from parental U937 and FS-induced cells at 8, 24 or 48 h of culture using TRIzol (Life Technologies, Gaithersburg, MD, USA). The RNA was processed and hybridized to human genome U95Av2 gene chips, following the manufacturer's protocols (Affymetrix, Santa Clara, CA, USA). Chips were scanned and analyzed using Microarray software (Affymetrix) as described previously.22 Upregulation of a gene in the FS-induced cells was determined by comparing its level of expression with that in the control U937 cells.

Real-time RT-PCR

Quantitative real-time RT-PCR (TaqMan) analysis was performed with an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The amplification mix included 100 ng RNA, 0.2 muM of each primer, 0.2 muM of each probe and TaqMan one-step RT-PCR master mix reagent in a total reaction volume of 40 mul. As an internal control, amplification of GAPDH was performed in the same reaction mix and detected with an alternatively labeled probe. Triplicates of each standard and sample were assessed. Primer sequences are available upon request.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% PBS–TritonX-100 and incubated with mouse anti-flag antibody (Sigma, St Louis, MO, USA) and Alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Cells were covered with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) and then analyzed with an Olympus BX-51 fluorescence microscope.

Western blot analysis

Total cell lysates were separated on 10% SDS-PAGE gels and transferred to polyvinylidene diflouride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with a solution of 5% low-fat milk in tris-buffered saline and 0.05% Tween 20 for 1 h. Samples were incubated with primary antibodies overnight at 4°C and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized with chemiluminescence (Amersham Biosciences, Little Chalfont, UK). To control for equal loading, membranes were either stained with fast-green before blocking or stripped with a solution containing 50 mM glycine and 150 mM sodium chloride (pH 1.8) and reprobed with anti-Actin antibody. The following antibodies were used for Western blotting: Actin, ERK1, ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38/MAPK, phospho-p38/MAPK (Cell Signaling Technology), active-MAPK (pTEpY) (Promega, Madison, WI, USA) and Flag (Sigma, St Louis, MO, USA).

STAT1 DNA-binding assay

DNA-binding activity of STAT1 was detected using the TransAM method following the manufacturer's protocols (Active Motif, Carlsbad, CA, USA). Briefly, active STAT1 complexes in nuclear extracts from FS-uninduced and FS-induced cells were captured by binding them to a consensus oligonucleotide that was immobilized in wells of a 96-well plate. A colorimetric reaction using anti-STAT1 primary antibody and HRP-conjugated secondary antibody was used to identify the bound protein. The colorimetric readout was quantified by spectrophotometry. Each sample and positive control was analyzed in triplicate.

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Results

SET and vitamin D3 stimulate the expression of different markers on U937 cells

We recently reported that tetracycline (tet)-regulatable overexpression of flag-tagged SET (FS) in U937 cells blocked G1/S transition and stimulated differentiation as detected by upregulation of the cell surface marker CD11b.21 Vitamin D3 stimulates monocytic differentiation in U937 cells via vitamin D3 receptor-dependent expression of surface markers CD11b and CD14.23 To determine whether overexpression of SET mimics other cellular responses to vitamin D3, we assessed CD14 expression in FS-induced (tet [-]) cells. A 4-day induction of FS stimulated the expression of CD11b in 76% of the cells but CD14 expression remained undetectable (Figure 1a), whereas addition of vitamin D3 activated the expression of CD14 and CD11b in over 75% of the cells, regardless of whether the cells were maintained in the presence (FS-uninduced) or absence of tet (FS-induced) (Figure 1b). These results suggest that overexpression of SET induces differentiation of U937 cells along a pathway distinct from that induced by vitamin D3.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

SET overexpression in U937 cells upregulates CD11b and CD86 and causes dendritic cell-like morphologic changes. (a) FS-expressing cells cultured for 4 days with (FS tet+) or without tetracycline (FS tet-) were analyzed by FACS for the expression of the cell surface markers CD14, CD11b and CD86. (b) In a parallel experiment, the same analysis was performed on cells cultured for 4 days with or without tetracycline in the presence of vitamin D3. Data in (a) and (b) represent the mean (plusminuss.d.) from three experiments. (c) Phase contrast images showing the morphological differences between FS-uninduced (FS tet+) and FS-induced (FS tet-) cells. The FS-induced cells show formation of pseudo-pods (arrows), whereas the FS-uninduced cells remain round.

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Ectopic SET stimulates the expression of calcium-responsive genes and dendritic cell (DC) markers

To determine the comprehensive profile of genes whose expression is induced by SET overexpression, we performed microarray analyses. We identified 18 genes that were significantly upregulated (two-fold or greater increase in expression) and eight genes that were significantly downregulated (two-fold or greater decrease in expression). Interestingly, six out of 18 upregulated genes (GTP-cyclohydroxylase I, protein kinase JNK2, myocyte specific enhancer factor 2A (MEF2A), Ins(1,3,4,5)P4-binding protein (IP4BP), lymphocyte-specific protein 1 (LSP1) and type 3 inositol 1,4,5-trisphosphate receptor (IP3R3) (Table 1)) are directly or indirectly regulated by calcium.24, 25, 26, 27, 28, 29


Expression levels of four of these six upregulated genes were confirmed using real-time RT-PCR analysis. At 8 and 24 h of FS induction, the level of SET expression in the cells in the absence of tet increased 2.9- and 5.5-fold, respectively, compared with that in parental U937 cells (Figure 2a). Although the microarray analysis showed that in these cells only LSP1 was upregulated more than two-fold after 24 h, real-time RT-PCR analysis showed that GTP-cyclohydroxylase I (Figure 2b) and IP4BP were upregulated more than two-fold after 8 h, and MEF2A was upregulated to that level at 24 h (Table 1).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

SET overexpression upregulates GTP cyclohydrolase I mRNA. Real time RT-PCR analysis performed to detect the levels of GTP cyclohydrolase I and SET mRNA using total RNA from FS-uninduced (FS tet+), FS-induced (FS tet-), and parental U937 cells at 8, 24 and 48 h of culture. All samples were analyzed in triplicate and histograms show the average amount of (a) SET mRNA and (b) GTP cyclohydrolase I. Levels of transcription were normalized by using GAPDH mRNA as an internal control. Data represent the mean of three experiments (plusminuss.d.).

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Given that SET stimulated the expression of Ca2+-regulated genes and elevation of the intracellular calcium level is involved in DC differentiation of human promyelocytic cells, peripheral blood monocytes, and chronic myelogenous leukemia progenitor cells,30, 31 we monitored the appearance of the DC markers CD80, CD83, CD86, and HLA-DR after FS induction by using FACS analysis. In addition to CD11b, 60% of FS-induced cells expressed CD86 at day 4 (Figure 1a) but failed to express other DC markers (data not shown). Nonetheless, the cells acquired a DC-like morphology (Figure 1c). Thus, the marker analysis suggested that the induction of the DC differentiation program was only partial. Moreover, vitamin D3 treatment overruled these effects of FS and drove the differentiation program toward a monocytic fate, as indicated by the expression of CD11b and CD14 but not CD86 in FS-induced cells (Figure 1b).

Inhibitors of calmodulin (CaM) and calmodulin-dependent kinase II (CaMKII) block SET-induced surface marker expression and morphologic changes

The Ca2+/CaM complex regulates calcium signaling through activation of target kinases such as CaMKII and phosphatases such as calcineurin. To assess the role of Ca2+-dependent signaling pathways in FS-stimulated DC differentiation, we treated FS-expressing cells with the Ca2+/CaM antagonist W-7, the calcineurin inhibitor cyclosporin A (CsA), or the CaMKII inhibitor KN-93. Incubation with W-7 reduced the level of CD11b expression 2.6-fold and that of CD86 by 3.2 fold (Figure 3a) while KN-93 treatment decreased these levels two- and four-fold, respectively (Figure 3c). Both inhibitors also reversed the FS-induced morphologic changes (Figure 3b). In contrast, CsA only moderately altered the level of FS-stimulated expression of surface markers (Figure 3c). Together, these results indicated that Ca2+/CaM and CaMKII activation is involved in FS-stimulated DC-like differentiation but probably there are also other contributing factors since the inhibition via Ca2+/CaM and CaMKII was not complete.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Inhibitors of, Ca2+/CaM, CaMKII or MAPK/ERK pathways inhibit FS-induced immunophenotypic and morphologic changes. (a) Ca2+/CaM inhibitor W-7 blocked FS-induced expression of CD11b (upper right panel, green) and CD 86 (lower right panel, red). The upper and lower left panels show expression of these markers in FS-uninduced (tet+) cells and middle panels in FS-induced (tet-) cells in absence of inhibitors. The percentage of cells expressing the marker is indicated in each panel. (b) Morphologic changes induced by FS overexpression were reversed by W-7, KN-93 (CaMKII inhibitor) and PD98059 ([PD] MEK1/ERK1,2 inhibitor). (c) PD98059 (PD) and KN-93 substantially reduced FS-induced expression of CD11b and CD86, whereas cyclosporine A (CsA) and SB202190 (SB), a specific inhibitor of p38, only moderately inhibited the expression of the markers. Data represent the average (plusminuss.d.) of triplicate experiments. (d) The total amount and the active p38/MAPK (p-p38) and MAPK/ERK1/ERK2 (pERK1 and pERK2) were determined by using Western blot analysis at indicated time points in FS expressing cells that were cultured with (tet+) or without (tet-) tetracycline. FS expression was detected with anti-Flag antibody, and equal loading was verified by anti-Actin antibody.

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The MAPK/ERK1/ERK2 pathway is required for SET-induced differentiation of U937 cells

Since CaMKII contributes to the FS-stimulated phenotype, it is noteworthy that CaMKII could provide a link between calcium signaling and MAPK/ERK activation through its regulation of the serine/threonine kinase RAF-1.32, 33, 34 Also, SET overexpression in NIH3T3 cells activates the MAPK/ERK pathway by inhibiting PP2A.17 In addition, both MAPK/ERK and p38/MAPK activation are involved in the maturation of human monocyte-derived DC.35 Thus, we next explored whether MAPK/ERK and p38MAPK activation plays a role in SET-induced differentiation by using highly selective inhibitors of MEK1/ERK (PD98059) and p38/MAPK (SB202190), respectively. PD98059 (PD) treatment partially reversed SET-induced morphologic changes (Figure 3b) and decreased the expression of CD11b and CD86 by 1.7- and 4.4-fold, respectively, (Figure 3c). Western blot analysis supported results from the PD98059 experiment and showed transient higher levels of phospho-ERK1 at 12 h and a higher level of phospho-ERK2 at 12 and 14 h of FS induction (Figure 3d). In contrast, the p38/MAPK inhibitor SB202190 (SB) did not block SET-stimulated expression of CD11b or CD86 (Figure 3c), although SET overexpression transiently activated p38/MAPK at 12 h, as revealed by increased phospho-Thr180/Tyr182p38MAPK (Figure 3d). The transient increase in phosphorylation of ERK1 and p38/MAPK followed a transient increase in protein levels (Figure 3d) in FS-induced cells, while phospho-ERK2 initially also followed the increase in ERK2 protein but became dephosphorylated after 16 h of induction. The increase in FS background levels (compare all tet[+] lanes in Figure 3d) are due to necessary washing of the cells at the beginning of the experiment (not shown; see Materials and methods). None of the inhibitors affected FS expression or the viability of the cells (data not shown). These results suggest that in addition to Ca2+ signaling and activation of CaMKII, SET-induced DC-like differentiation also requires activation of MAPK/ERK1/2.

SET overexpression partially mimics but does not substitute interferon-bold italic gamma signaling

The changes induced by ectopic SET expression mentioned above share similarities to those found in IFN-italic gamma-treated cells.36, 37, 38, 39, 40, 41 Since the signal transducing activator of transcription-1 (STAT1) moderates IFN-italic gamma-induced gene expression,42 we analyzed whether ectopic expression of SET activates STAT1 DNA binding, which is a function of its phosphorylation at tyrosine (Y701). Western blot analysis after 8, 11, 12, 14, 16 and 24 h of FS induction showed no Y701 phosphorylation of STAT1 and ELISA-based transcription factor-binding assays showed no difference in the DNA-binding activity of STAT1 after FS induction at the same time points (data not shown). Thus, SET induction does not fully mimic IFN-italic gamma signaling.

Finally, we analyzed whether endogenous SET expression is affected by IFN-italic gamma stimulation. Real-time RT-PCR analysis of SET mRNA in parental U937 cells showed that IFN-italic gamma treatment upregulated SET transcription in a dose-dependent manner (Figure 4a). Timecourse experiments showed that the amount of SET mRNA returned to basal levels 2 h after IFN-italic gamma treatment (Figure 4b). This suggests that SET expression might be subject to IFN-italic gamma signaling.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Interferon-italic gamma treatment of U937 cells transiently induces expression of endogenous SET. (a) The amount of SET mRNA in RNA samples from U937 cells treated for 1 h with the indicated concentrations of human recombinant INF-italic gamma (InterMune, Inc., Brisbane, CA) was determined by real-time RT-PCR. (b) Time course of SET mRNA expression, as determined by real-time RT-PCR analysis in untreated cells and during treatment with the optimum dose of INF-italic gamma (250 U/ml). Levels of transcription in (a) and (b) were normalized by using GAPDH mRNA as an internal control.

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Discussion

Ectopic expression of SET in U937 cells induces differentiation towards a DC phenotype and SET-stimulated acquisition of DC-like features requires calcium signaling, CaMKII and MAPK/ERK1/ERK2 activation. U937 cells that overexpress SET undergo a number of changes similar to cells treated with IFN-italic gamma, that is, they exit the cell cycle;36 upregulate the expression of GTP cyclohydrolase I,37 CD11b39 and CD86;38 and activate the calcium signaling/CaMKII40 and MAPK/ERK pathways,41 but fail to activate STAT1, the main mediator of IFN-italic gamma signaling.42

IFN-italic gamma signaling orchestrates different cellular programs, including antimicrobial and antitumor mechanisms as well as cell proliferation and differentiation, through transcriptional regulation of target genes.43 In response to IFN-italic gamma, STAT1 becomes phosphorylated on Y701, and STAT1 dimers migrate into the nucleus and activate the expression of genes that contain STAT1-binding sites.44 Given the lack of Y701 phosphorylation and the failure to induce STAT1 DNA binding, we conclude that, despite the many similarities, SET induction cannot fully mimic IFN-italic gamma stimulation. Therefore, the upregulation of CD86 expression, a direct transcriptional target of STAT1,38 cannot be mediated by STAT1 in our SET-overexpressing cells. However, CD86 is also a direct target of NFkappaB,38 a transcription factor that is activated by Ca2+ flux45, and therefore, a good candidate to upregulate this gene. Active NFkappaB is also necessary for CD11b expression.46 Despite the differences in STAT1 activation after IFN-italic gamma treatment or SET overexpression, it is reasonable to speculate that SET induction is part of the IFN-italic gamma response, given the transient upregulation of SET after treatment of U937 cells with IFN-italic gamma (Figure 4b).

The well-known inhibitory effect of SET on PP2A10, 12, 14 should at least in part account for the observed activation of MAPK/ERK and CaMKII since PP2A negatively affects MAPK/ERK and CaMKII activation by dephosphorylation.17, 47 Therefore, the differentiation response of FS-induced U937 cells could be caused by inhibition of PP2A and activated calcium signaling which is induced via many stimuli such as stress or receptor activation.48

Currently, we do not know how SET expression activates calcium signaling. However, an integral role of SET in the stress response, as indicated by transient activation of p38/MAPK, may provide some clues, despite the observation that SB202190, the inhibitor of p38/MAPK, had no effect on differentiation. Also, there are many similarities between stress responses and the response to IFN-italic gamma treatment such as induction of calcium flux and MAPK/ERK activation.41

In FS-induced U937 cells, the activation of p38 and ERK/MAPKs are triggered soon after SET induction, when SET is only moderately upregulated. In fact, high levels of SET appear to inhibit p38 and ERK/MAPKs phosphorylation as well as protein levels (Figure 3d). This finding supports the physiological significance of the response and indicates that it is not simply a side effect of massive overexpression of SET. Also during IFN-italic gamma stimulation, endogenous SET is induced to only a moderate increase. A slightly confounding observation is that the level of FS also increases in cells that remain under tet repression. This is caused by the obligatory washing of the cells at the beginning of the experiment to ensure that both tet[+] and tet[-] cells were handled in the same way. We believe that this upregulation remains below a level that induces differentiation and phosphorylation of ERK1,2 and p38/MAPK, whereas FS induction in tet[-] cells reaches a level high enough to induce these changes. Indeed, we never detected any signs of differentiation in tet[+] cells.

Recently, it was reported that SET downregulation rather than upregulation was associated with the induction of differentiation in the bladder carcinoma cell line TSU-Pr1.49 Although these data seem contradictory to our findings the two data sets are difficult to compare because the authors did not assess the effect of SET overexpression in TSU-Pr1 cells. In addition, the effects of SET might be different in different cell types.

In addition to calcium signaling-mediated transcriptional effects, SET overexpression might also affect the activity of the HuR complex of which it is a component.9 HuR regulates stabilization of mRNAs containing AU-rich elements (ARE) in their 3'UTRs,50, 51 which targets them for rapid degradation.52, 53 Elevated SET expression might, therefore, increase the activity of the HuR complex and inhibit degradation of short-lived mRNAs, which may encode proteins important for differentiation. Future studies will address this possibility. To assess this and the role of SET in stress responses, we are beginning to analyze the effects of a SET-knockout mutation in mice.

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Acknowledgements

We thank Dr Youngsoo Lee for expert help with the microarray data analysis and real time RT-PCR experiments, Dr T Copeland for SET antibody, Drs Ann-Mary Hamilton-Easton and Richard Ashmun for FACS analysis, Dr Susan Magdaleno for light microscopy and Charlette Hill for secretarial assistance. This work was supported by NIH Grant CA-76480-05, Cancer Center Support CA-21765 and by the American Lebanese Syrian Associated Charities (ALSAC).

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